Integration*of*Methods*to*Study*Mate*Choice*Behavior*in*Treefrogs*

*

*

A*Dissertation*

Presented*to*

The*Faculty*of*the*Graduate*School*

At*the*University*of*Missouri*

************* *

*

In*Partial*Fulfillment*

Of*the*Requirements*for*the*Degree*

Doctor*of*Philosophy*

* *

By*

JENNIFER*JEAN*HENDERSON*

Dr.*H.*Carl*Gerhardt,*Dissertation*Supervisor* *

DECEMBER**2014*

* * *

* The*undersigned,*appointed*by*the*dean*of*the*Graduate*School,*

have*examined*the*dissertation*entitled*

INTEGRATION*OF*METHODS*TO*STUDY*MATE*CHOICE*BEHAVIOR*IN*TREEFROGS*

Presented*by*Jennifer*Jean*Henderson*

A*candidate*for*the*degree*of**

Doctor*of*Philosophy*

And*hereby*certify*that,*in*their*opinion,*it*is*worthy*of*acceptance.*

*

*

* Professor*H.*Carl*Gerhardt*

*

* Professor*Lori*Eggert*

*

* Professor*David*Geary*

*

* Professor*Johannes*Schul*

*

*

*

* *

* *

*

*

“If*we*knew*what*it*was*we*were*doing,**

it*would*not*be*called*research,*would*it?”*

XAlbert*Einstein*

*

*

*

I*finally*figured*out*what*I*was*doing.*

*

*

*

And*yes,*now*I*am*done*with*school.* *

ACKNOWLEDGEMENTS.

My*path*to*this*point*started*by*responding*to*a*flyer*seeking*undergraduate* researchers*to*study*behavior*of*treefrogs*at*the*University*of*Minnesota.*After*two* summers*spent*working*for*Mark*Bee,*I*decided*to*continue*on*to*graduate*school*and* continue*researching*frogs*with*my*advisor,*Carl*Gerhardt.*The*summer*before*I*started,*

Mark*came*back*from*the*Animal*Behavior*conference*where*he*and*Carl*were* roommates.*He*told*me*that*he*and*Carl*had*so*much*fun*and*that*they*had*planned*out* my*life*for*me.*I*was*to*study*the*Cope’s*gray*treefrog*and*I*would*research*their* morphological,*behavioral,*and*genetic*differences.*Being*22*years*old*and*had*not* spent*a*single*day*in*graduate*school,*I*laughed*because*I*was*going*to*do*whatever*I* wanted.*Fast*forward*five*and*half*years*later,*here*I*am*looking*at*my*dissertation’s* table*of*contents,*specifically*Chapter*4.*Well*played,*gentlemen,*well*played.*

Beyond*Mark*and*Carl,*who*apparently*played*the*long*game*in*terms*of* bringing*me*to*where*I*am*now,*I*have*to*thank*my*committee*members.*Walking*into*

Lori*Eggert’s*lab*my*experience*consisted*of*knowing*a*micropipette*had*two*stops*on* the*plunger.*Walking*out*of*her*lab,*I*know*enough*to*be*a*competent*geneticist.*Lori* opened*an*entirely*new*field*for*me.*Johannes*Schul*straight*up*helped*me*become*a* better*scientist.*My*understanding*of*experimental*design,*acoustics,*and*proximate*and* ultimate*mechanisms*is*largely*due*to*him.*David*Geary*made*me*think*of*my*research*

ii** * questions*from*an*entirely*new*point*of*view.*Dave*often*asked*questions*that*I*hadn’t* thought*of*myself*and*I*think*my*research*is*stronger*because*of*it.*

I*had*a*lot*of*help*along*the*way.*To*complete*chapters*two*and*three,*Gary*Rose* from*the*University*of*Utah*was*kind*enough*to*give*me*his*insights*on*what*might*be* going*on*in*the*brain*to*help*me*make*sense*of*my*results.*For*chapter*four,*Aaron*

Bossert*valiantly*helped*me*locate*the*elusive*populations*of*Hyla%chrysoscelis*from*the* western*part*of*Missouri.*Chapter*four*would*have*never*been*completed*had*it*not* been*for*the*help*of*Bill*Peterman,*Sheena*Feist,*Tabitha*Finch,*and*Emily*Puckett.*Like*I* said,*I*knew*next*to*nothing*about*genetics.*I*know*a*bunch*of*stuff*now*thanks*to*them.*

My*lab*mates,*past*and*present,*Sarah*Humfeld,*Katy*Klymus,*Michael*Reichert,*Jessica*

Merricks,*and*Mitch*Tucker,*made*my*experience*both*educational*and*fun.*Mostly*fun.*

We*elevated*the*department’s*holiday*door*decorating*contest*to*a*brand*new*level.*I* have*probably*eaten*my*weight*in*Twix*bars*and*Reese’s*peanut*butter*cups*thanks*to*

Nila*Emerich.*I*also*have*considerably*less*frequent*food*cravings*now*that*I’m*not*in*

Nila’s*office*everyday*talking*about*cooking.*Debbie*Allen*was*also*a*frequent*snack* provider*and*my*biggest*cheerleader*in*the*Life*Sciences*Fellowship*program.*Deanna*

Lankford*and*Anna*Waldron*opened*up*a*lot*of*doors*for*me*in*the*world*of*science* outreach.*I*had*so*many*opportunities*to*develop*my*skills*in*this*area.*They*also* brought*me*to*Susan*Renoe,*and*I*am*entirely*convinced*is*the*reason*why*I*have*such*a* wonderful*job*to*start*me*off*in*the*real*world.*In*my*time*as*a*graduate*student,*I*am* grateful*to*have*been*welcomed*into*the*Disney*movie*crew:*Tabitha*Finch,*Brandie*

Morgan,*Jessica*Merricks,*Katy*Klymus,*and*Katie*Kilmer.*They*were*there*from*the* iii** * beginning*and*I*probably*wouldn’t*have*made*it*through*graduate*school*without*them.*

I*also*wouldn’t*have*made*it*out*of*graduate*school*without*Katie*O’Donnell,*Britt*

Ousterhout,*Daniel*Godwin,*Emily*Puckett,*Chris*Kassotis,*Freya*Rowland,*Tom*

Anderson,*and*Alice*Tipton.*

Last*but*not*least,*I*owe*a*lot*to*my*family.*I*don’t*think*my*parents*ever* questioned*what*I*wanted*to*do.*They*might*not*have*understood*it*all*but*that*didn’t* stop*them*from*supporting*me*completely.*I*don’t*think*I*could*have*had*a*better* environment*to*shape*me*as*a*person.*They*had*that*amazing*mix*of*setting*me*free*to* find*my*own*way*and*figure*it*out*for*myself,*but*at*the*same*time,*I*always*knew*they’d* be*right*behind*me*if*I*needed*help.*They’re*probably*prouder*of*this*accomplishment* than*I*am.*

*

I*can*probably*also*say*now*that*I’m*sorry,*Mom,*for*going*out*to*the*frog*ponds*alone* at*night.*I*did*it*more*often*than*I*admitted*to*you.*But*I*have*a*PhD*now,*so*I*don’t*think* you*can*get*that*mad*about*it.*Besides,*I’m*still*alive,*so*it*obviously*turned*out*fine.

iv** * *

TABLE.OF.CONTENTS.

Acknowledgements*...... *ii*

List*of*Tables*...... *vii*

List*of*Figures*...... *viii*

Chapter*1.*Introduction*...... *1*

Literature*Cited*...... *6*

Chapter*2.*Restoration*of*call*attractiveness*by*novel*acoustic*appendages*in*grey* treefrogs*...... *8*

Introduction*...... *10*

Methods*...... *13*

Results*...... *18*

Discussion*...... *21*

Literature*Cited*...... *25*

Chapter*3.*Effect*of*temporal*properties*on*attractiveness*of*novel*complex*calls*in*the* cope’s*grey*treefrog,*hyla%chrysoscelis*...... *38*

Introduction*...... *40*

Methods*...... *43*

Results*...... *46*

Discussion*...... *48*

Literature*Cited*...... *54*

Chapter*4.*Genetic%Differentiation*of*Populations*of*the*Cope's*Grey*Treefrog,*Hyla% chrysoscelis*...... *61*

Introduction*...... *62*

ii** * Methods*...... *65*

Results*...... *67*

Discussion*...... *68*

Literature*Cited*...... *73*

Chapter*5.*Conclusions*...... *85*

Literature*Cited*...... *90*

Vita*...... *92*

iii** * LIST.OF.TABLES.

TABLE...... PAGE.

Supplementary*Table*2X1.*Choices*of*females*in*individual*behavioral*tests*...... *36*

Table*4X1.Primers*for*microsatellite*loci*in*Hyla%chrysoscelis*...... *77*

Table*4X2.Population*genetic*summary*statistics*...... *78*

Table*4X3.*Pairwise*FST*for*all*21*population*pairs*...... *79*

Table*4X4.*Pairwise*FST*for*population*pairs*using*clusters*identified*by*STRUCTURE*...... *80*

Supplementary*Table*4X5.*Results*of*Evanno*test*best*supporting*three*genetic*clusters *...... *81*

*

*

*

* *

vii* * * LIST.OF.FIGURES.

FIGURE...... PAGE.

Figure*2X1.*Examples*of*stimuli*used*...... *27*

Figure*2X2.*Discrimination*against*calls*with*gaps*(one*missing*pulse)*...... *28*

Figure*2X3.*Effect*of*relative*intensity*of*the*appendage*...... *30*

Figure*2X4.*Effect*of*appendage*duration*...... *31*

Figure*2X5.*Effect*of*gap*location*...... *32*

Figure*2X6.*Effect*of*temporal*order*...... *33*

Figure*2X7.*Discrimination*against*calls*with*one*or*three*abnormally*short*pulses*...... *34*

Figure*2X8.*Effect*of*appendages*in*restoring*attractiveness*of*call*with*suboptimal* pulses*...... *35*

Figure*3X1.*Examples*of*calls*used*...... *57*

Figure*3X2.*Proportion*of*females*choosing*each*alternative*when*calls*contained*gaps58*

Figure*3X3.*Choices*of*females*in*tests*of*a*standard*advertisement*call*versus*an* alternative*...... *59*

Figure*3X4.*Temporal*order*of*the*appendage*relative*to*the*pulsed*portion*of*the*call* affected*attractiveness*...... *60*

Figure*3X5.*The*proportion*of*females*choosing*each*alternative*in*a*direct*test*of*a*call* containing*either*a*following*or*leading*appendage*...... *61*

Figure*3X6.*When*the*silent*interval*between*the*pulsed*portion*and*the*appendage*was* reduced*to*20*ms,*the*restoration*effects*at*50*ms*were*reversed*...... *62*

Figure*4X1.*Locations*of*samples*collected*...... *82*

Figure*4X2.*Results*of*genetic*clustering*in*STRUCTURE*for*Hyla%chrysoscelis*individuals* sampled*across*the*range*at*two*and*three*genetic*clusters*(K)*...... *83*

Figure*4X3.*Geographic*distributions*of*genetic*clusters*...... *84*

viii* * * CHAPTER.1.

INTRODUCTION.

Jennifer*J.*Henderson*

Division*of*Biological*Sciences,*University*of*Missouri,*Columbia,*MO*65211

1** * Niko*Tinbergen*posed*the*central*question*of*ethology,*“Why*do*animals*behave* the*way*that*they*do?”**To*answer*such*a*broad*question,*Tinbergen*proposed*that* behavior*should*be*examined*through*four*levels*of*analysis:*function,*evolution,* mechanism,*and*development*(Tinbergen,*1963).*This*integrative*approach*would*result* in*a*more*thorough*and*complete*understanding*of*the*actions*of*animals.**

Anurans*are*a*widely*studied*model*organism*for*studying*Tinbergen’s*(1963)* four*levels,*and*much*is*known*about*their*behaviors.*Typically,*males*aggregate*in* dense*choruses;*females*select*their*mate*based*on*the*male’s*call*(Gerhardt*&*Huber,*

2002).*In*these*choruses,*males*may*be*in*close*proximity*with*calling*rivals*for*potential* female*mates.*For*many*species,*males*have*two*distinct*calls*types,*an*aggressive*call* that*is*typically*unattractive*to*females*and*an*attractive*advertisement*call.*Male*frogs* of*species*that*defend*territories*over*a*large*part*of*the*breeding*season*will*habituate* to*calling*neighbors*and*only*increase*in*aggressive*call*frequency*when*presented*with* a*call*from*a*novel*competitor*(Bee*&*Gerhardt,*2001;*Marshall*et%al.,*2003;*Owen*&*

Perrill,*1998).*At*the*mechanistic*level,*this*behavioral*adaptation*correlates*to* habituation*and*decreased*firing*of*neurons*to*repetitive*acoustic*stimuli*(Megela*&*

Capranica,*1983).*Female*frogs*must*also*respond*appropriately*to*signals*by*localizing* an*attractive*call.*In*some*species,*pulse*rate*of*a*male’s*call*alone*is*attractive*and*can* be*sufficient*for*females*to*select*conspecific*males*(Gerhardt*&*Huber,*2002).*Neurons* in*the*auditory*midbrain*that*are*tuned*to*narrow*ranges*of*amplitude*modulation*

(pulse*rate)*may*be*a*possible*neural*mechanism*for*this*behavioral*selectivity*(Feng*et% al.,*1990;*Wilczynski*&*Capranica,*1984).**

2** * In*multiple*anuran*species,*including*the*gray*treefrog*Hyla%versicolor,*neurons* that*are*selective*for*temporal*patterns*have*been*identified*(Edwards*et%al.,*2002).*

Behavioral*selectivity*has*also*been*shown*to*correlate*with*the*response*properties*of* these*neurons*(Schwartz*et%al.,*2010).*In*chapter*2,*my*aim*was*to*examine*how* temporally*selective*neurons*influence*female*behavioral*responses*to*novel*acoustic* appendages*to*typical*male*calls.*When*presented*with*novel*complex*calls,*females* only*responded*to*calls*with*appendages*that*followed*the*natural*pulsed*portion*of*the* call*(Gerhardt*et%al.,*2007).*Based*on*the*response*properties*of*the*temporally*selective* neurons,*I*created*unattractive*calls*by*adding*gaps*in*the*pulse*train*and*examined*the* effect*of*a*variety*of*appendage*types*on*call*attractiveness.*

For*chapter*3,*I*did*a*similar*experiment*with*the*closely*related*Cope’s*gray* treefrog,*Hyla%chrysoscelis.*Unlike*H.%versicolor%females*which*use*pulse*duration,*shape,* and*interval*for*recognition,*H.%chrysoscelis*females*rely*on*pulse*rate*alone*(Schul*&*

Bush,*2002).*Differences*between*closely*related*species*in*preference,*as*well*as* recognition*mechanism*for*the*preference,*have*been*identified*in*multiple*taxa*

(Barbosa*et%al.,*2006;*Couldridge*&*Alexander,*2002;*Hennig,*2003;*Schul,*1998;*Wong*et% al.,*2005).*Since*previous*experiments*showed*that*both*leading*and*following* appendages*could*be*attractive*to*H.%chrysoscelis*females,*I*further*explored*the* differences*in*behavioral*response*to*novel*complex*calls*between*H.%versicolor*and*H.% chrysoscelis.**

While*I*examined*behavioral*differences*between*closely*related*species*in* chapters*2*and*3,*it*was*behavioral*differences*observed*within*the*single*species*H.%

3** * chrysoscelis*that*provided*the*impetus*for*the*research*described*in*chapter*4.*Females* from*Minnesota*preferred*novel*elements*at*the*beginning*of*the*call,*while*females*in*

Missouri*did*not*(Gerhardt*et*al.,*2007;*Seeba*et%al.,*2010).*Additionally,*H.%chrysoscelis* from*populations*in*Minnesota*and*Missouri*differed*in*preference*for*stimuli*that*only* contained*one*of*the*advertisement*call’s*two*frequency*peaks*(Gerhardt*et%al.,*2007;*

M.A.*Bee,*unpublished*data).*Previous*work*using*multiple*genetic*methods*have* delineated*H.%chrysoscelis*into*two*distinct*lineages,*one*in*the*eastern*part*of*the*range*

(including*populations*from*Missouri)*and*one*in*the*western*area*(including* populations*from*Minnesota)*(Holloway*et%al.,*2006;*Ptacek*et%al.,*1994).*Anecdotally,* frogs*from*Minnesota*are*typically*smaller*in*size,*bright*green,*and*smoothXskinned* while*frogs*from*central*and*southeastern*Missouri*frogs*are*greyXgreen,*large*in*size,* and*have*bumpy*skin*(J.*Henderson,*personal*observation).*

In*chapter*4*I*used*eight*microsatellite*loci*to*determine*population* differentiation*across*the*geographic*range*of*H.%chrysoscelis.%It*is*estimated*that* lineages*of*H.%chrysoscelis*diverged*558,000*years*ago*(Ralin*et%al.,*1983),*and*thus,* microsatellites*are*wellXsuited*for*this*investigation.*Microsatellites,*which*are*a*series* of*repeat*sequences,*mutate*frequently*during*DNA*replication;*these*high*mutation* rates*make*microsatellites*a*useful*tool*for*studies*on*ecological*time*scales*(Eisen,*

1999;*Schlötterer,*2000).*The*fineXscale*population*structure*of*H.%chrysoscelis*can* provide*a*muchXneeded*backbone*for*future*work*correlating*behavioral*and*genetic* differentiation*and*the*identification*of*contact*zones*between*lineages.

4** * LITERATURE(CITED(

Barbosa,*D.,*Font,*E.,*Desfilis,*E.,*&*Carretero,*M.*(2006).*Chemically*Mediated*Species* Recognition*in*Closely*Related*Podarcis*Wall*Lizards.*Journal%of%Chemical%Ecology,* 32(7),*1587–1598.*

Bee,*M.*A.,*&*Gerhardt,*H.*C.*(2001).*Habituation*as*a*mechanism*of*reduced*aggression* between*neighboring*territorial*male*bullfrogs*(Rana%catesbeiana).*Journal%of% Comparative%Psychology.*US:*American*Psychological*Association.**

Couldridge,*V.*C.*K.,*&*Alexander,*G.*J.*(2002).*Color*patterns*and*species*recognition*in* four*closely*related*species*of*Lake*Malawi*cichlid.*Behavioral%Ecology%,*13%(1*),*59– 64.*

Edwards,*C.*J.,*Alder,*T.*B.,*&*Rose,*G.*J.*(2002).*Auditory*midbrain*neurons*that*count.* Nat%Neurosci,*5(10),*934–936.*

Eisen,*J.*(1999).*Mechanistic*basis*for*microsatellite*instability.*In*D.*B.*Goldstein*&*C.* Schlötterer*(Eds.),*Microsatellites:%Evolution%&%Approaches*(pp.*197–212).*Oxford,* UK:*Oxford*University*Press.*

Feng,*A.*S.,*Hall,*J.*C.,*&*Gooler,*D.*M.*(1990).*Neural*basis*of*sound*pattern*recognition* in*anurans.*Progress%in%Neurobiology,*34(4),*313–329.*

Gerhardt,*H.*C.,*&*Huber,*F.*(2002).*Acoustic%Communication%in%Insects%and%Anurans.* Chicago:*The*University*of*Chicago*Press.*

Gerhardt,*H.*C.,*Humfeld,*S.*C.,*&*Marshall,*V.*T.*(2007).*Temporal*order*and*the* evolution*of*complex*acoustic*signals.*Proceedings%of%the%Royal%Society%B:%Biological% Sciences,*274(1619),*1789–1794.*

Gerhardt,*H.*C.,*MartínezXRivera,*C.*C.,*Schwartz,*J.*J.,*Marshall,*V.*T.,*&*Murphy,*C.*G.* (2007).*Preferences*based*on*spectral*differences*in*acoustic*signals*in*four*species* of*treefrogs*(Anura:*Hylidae).*J%Exp%Biol,*210(17),*2990–2998.**

Hennig,*R.*M.*(2003).*Acoustic*feature*extraction*by*crossXcorrelation*in*crickets?* Journal%of%Comparative%Physiology%A:%Sensory,%Neural,%and%Behavioral%Physiology,* 189(8),*589–598.*

Holloway,*A.*K.,*Cannatella,*D.*C.,*Gerhardt,*H.*C.,*&*Hillis,*D.*M.*(2006).*Polyploids*with* different*origins*and*ancestors*form*a*single*sexual*polyploid*species.*The%American% Naturalist,*167(4),*E88–101.* 5** * Marshall,*V.*T.,*Humfeld,*S.*C.,*&*Bee,*M.*A.*(2003).*Plasticity*of*aggressive*signalling*and* its*evolution*in*male*spring*peepers,*Pseudacris*crucifer.*Animal%Behaviour,*65(6),* 1223–1234.*

Megela,*A.,*&*Capranica,*R.*(1983).*A*neural*and*behavioral*study*of*auditory* habituation*in*the*bullfrog,Rana*catesbeiana.*Journal%of%Comparative%Physiology,* 151(4),*423–434.*

Owen,*P.*C.,*&*Perrill,*S.*A.*(1998).*Habituation*in*the*green*frog,*Rana*clamitans.* Behavioral%Ecology%and%Sociobiology,*44(3),*209–213.*

Ptacek,*M.*B.,*Gerhardt,*H.*C.,*&*Sage,*R.*D.*(1994).*Speciation*by*polyploidy*in*treefrogs:* multiple*origins*of*the*tetraploid,*Hyla*versicolor.*Evolution,*48(3),*898–908.**

Ralin,*D.*B.,*Romano,*M.*A.,*&*Kilpatrick,*C.*W.*(1983).*The*tetraploid*treefrog*Hyla* versicolor:*evidence*for*a*single*origin*from*the*diploid*H.*chrysoscelis.* Herpetologica,*39(3),*212–225.*

Schlötterer,*C.*(2000).*Evolutionary*dynamics*of*microsatellite*DNA.*Chromosoma,* 109(365X371).*

Schul,*J.*(1998).*Song*recognition*by*temporal*cues*in*a*group*of*closely*related* bushcricket*species*(genus*Tettigonia).*Journal%of%Comparative%Physiology%A,*183(3),* 401–410.*

Schul,*J.,*&*Bush,*S.*L.*(2002).*NonXparallel*coevolution*of*sender*and*receiver*in*the* acoustic*communication*system*of*treefrogs.*Proceedings%of%the%Royal%Society%B:% Biological%Sciences,*269(1502),*1847–1852.*

Schwartz,*J.*J.,*Huth,*K.,*Hunce,*R.,*&*Lentine,*B.*(2010).*Effect*of*anomalous*pulse*timing* on*call*discrimination*by*females*of*the*gray*treefrog*(Hyla%versicolor):*behavioral* correlates*of*neurobiology.*J%Exp%Biol,*213(12),*2066–2072.*

Seeba,*F.,*Schwartz,*J.*J.,*&*Bee,*M.*A.*(2010).*Testing*an*auditory*illusion*in*frogs:* Perceptual*restoration*or*sensory*bias?*Anim%Behav,*79(6),*1317–1328.**

Tinbergen,*N.*(1963).*On*aims*and*methods*of*Ethology.*Zeitschrift%Für%Tierpsychologie,* 20(4),*410–433.*

Wilczynski,*W.,*&*Capranica,*R.*R.*(1984).*The*auditory*system*of*anuran*amphibians.* Progress%in%Neurobiology,*22(1),*1–38.*

6** * Wong,*B.*B.*M.,*Fisher,*H.*S.,*&*Rosenthal,*G.*G.*(2005).*Species*recognition*by*male* swordtails*via*chemical*cues.*Behavioral%Ecology,*16(4),*818–822.

7** * CHAPTER.2..

RESTORATION.OF.CALL.ATTRACTIVENESS.BY.NOVEL.ACOUSTIC. APPENDAGES.IN.GREY.TREEFROGS..

Jennifer*J.*Henderson*and*H.*Carl*Gerhardt*

Division*of*Biological*Sciences,*University*of*Missouri,*Columbia,*MO*65211*

*

* *

8** * INTRODUCTION(

Acoustic*communication*plays*a*prominent*role*in*mate*choice*in*many*kinds*of* animals,*and*acoustic*signals*may*convey*information*about*the*species,*physical* condition,*and*heritable*fitness*of*a*prospective*mate.*However,*not*all*preferences*are* based*on*such*properties*and*strong*responses*to*exaggerated,*supernormal*stimuli* have*been*observed*since*the*early*days*of*ethology*(Tinbergen*1951;*Andersson*1994).*

Formal*explanations*based*on*preXexisting*sensory*biases*(Ryan*&*Rand*1993)*or*hidden* preferences*(Arak*&*Enquist*1993)*have*received*much*attention*and*some*empirical* support.*Such*biases*may*contribute*to*the*evolution*of*complex*signals.**

Whereas*most*species*of*frogs*and*toads*produce*calls*consisting*of*a*single* acoustic*element*that*is*repeated,*some*exceptional*species,*scattered*throughout*a* wide*range*of*taxonomic*groups,*produce*calls*with*multiple*acoustic*elements*

(Gerhardt*&*Huber*2002).*In*two*groups*of*anurans,*experiments*have*shown*that* adding*elements*of*the*calls*of*males*of*another*species*to*the*singleXelement*speciesX typical*male*call*can*result*in*a*novel*complex*call*that*is*preferred*by*females*over*the* simple*normal*call*(Ryan*&*Rand*1993;*Gerhardt*et*al.*2007).*Whereas*these* experimental*results*support*the*idea*that*preXexisting*biases*may*underlie*the* evolution*of*complex*calls,*several*questions*arise*about*the*specific*sensory* mechanisms.*Are*complex*calls*more*attractive*simply*by*virtue*of*the*greater*duration* of*sensory*stimulation?**If*not,*then*what*are*the*characteristics*of*novel*signals*that* make*them*more*attractive*than*conspecific*signals?**Are*these*characteristics*common* to*a*wide*variety*of*taxa*or*specific*to*a*few*exceptional*species?**To*the*extent*that*a* 9** * widespread*set*of*criteria*exists,*are*there*also*common,*underlying*sensory* mechanisms*or*constraints?*

Studies*of*grey*treefrogs*(Hyla%versicolor*and*H.%chrysoscelis),*in*which*males* produce*simple*trills,*showed*that*adding*a*novel*element*to*the*trill*usually*enhanced* the*attractiveness*of*the*resulting*complex*call*relative*to*the*trill*alone*but*only*if*the* novel*element*followed*the*trill*in*time*(Gerhardt*et*al.*2007).*Subsequent*research*has* corroborated*this*temporalXorder*effect*in*H.%versicolor*over*a*wide*range*of*values*of* duration*of*the*trill*and*appendages*(novel*acoustic*elements)*and*magnitudes*of*the* silent*gap*between*elements*(Gerhardt*&*Humfeld,*in*prep).*By*contrast,*the*order* effect*was*weaker*in*H.%chrysoscelis,*and*given*short*silent*gaps,*complex*calls*with* leading*appendages*often*had*enhanced*attractiveness*(see*also*Seeba*et*al.*2010).*

Despite*their*close*relationship,*such*differences*are*not*entirely*surprising*because* females*of*the*two*species*differ*significantly*in*the*fineXscale*temporal*criteria*by*which* they*recognise*conspecific*trills*(Schul*&*Bush*2002).*

Our*goal*was*to*relate*the*behavioural*effectiveness*of*different*variants*of* novel*complex*calls*to*the*response*properties*of*a*class*of*temporally*selective*auditory* neurons*found*in*the*midbrain*and*characterized*in*several*species*of*frogs.*Our*data* are*derived*from*experiments*with*H.%versicolor,*the*greyXtreefrog*species*in*which*the* duration,*shape,*and*silent*interpulse*interval*all*play*a*role*in*species*recognition*(Schul*

&*Bush*2002).*Our*experiments*were*framed*in*part*by*descriptions*of*a*class*of* temporally*selective*neurons*(intervalXcounting*neurons),*identified*in*the*torus* semicircularis*of*several*anuran*species*(Adler*&*Rose*1998;*Edwards*et*al.*2002).*The* 10* * * torus*semicircularis,*a*homolog*of*the*inferior*colliculus*of*higher*vertebrates,*is*a*key* point*in*the*central*auditory*system,*integrating*forebrain*inputs,*acting*as*a*transition* between*the*brainstem*and*forebrain,*and*interfacing*with*the*motor*system*

(Wilczynski*&*Endepols*2006).*Thus,*the*response*properties*of*neurons*in*this*part*of* the*brain*are*almost*certainly*important*for*selective*phonotaxis*(orientation*and* movement*towards*sound).**

These*neurons*have*the*property*of*pulseXperiod*selectivity,*with*the*proviso* that*they*fire*only*after*a*threshold*number*of*correct*pulse*periods*(reciprocal*of*pulse* rate).*An*individual*pulse*elicits*small*excitation*followed*by*inhibition.*A*series*of*pulses* presented*with*optimal*pulse*periods*result*in*enhanced*excitation*that*overcomes* inhibition*and*results*in*spikes.*Pulse*periods*that*are*too*short*or*too*long*in*duration* reset*the*counting*process.*If*the*interval*between*pulses*is*increased,*continued* inhibition*and*a*decrease*in*excitation*result*in*neuronal*resetting*and*the*absence*of* spikes*(Edwards*et*al.*2007).*In*H.%versicolor,*behavioural*experiments*show*that*pulse* duration*must*also*exceed*some*minimum*value*and*that*interXpulse*silent*periods*(and* hence*pulse*duty*cycle)*can*then*vary*over*a*wider*range*than*found*in*natural*calls*

(Schul*and*Bush*2002).*Thus*in*addition*to*testing*signals*with*an*abnormally*long*pulse* period,*we*assessed*the*effects*of*shortening*the*duration*of*only*one*or*three*pulses*in* a*trill.*

Our*main*goal*was*to*test*the*hypothesis*that*following*appendages*in*grey* treefrogs*are*effective*because*once*intervalXcounting*neurons*integrate*and*fire*in* response*to*a*threshold*number*of*pulses*with*speciesXtypical*duration*and*period,*the* 11* * * majority*of*these*cells*will*continue*to*respond*tonically*throughout*the*duration*of*the* stimulus*(Adler*&*Rose*2000).*This*property*might*ensure*that*almost*any*acoustic* element*with*the*appropriate*spectral*composition*and*amplitude*could*be*effective*in* maintaining*stimulation*of*these*cells,*thus*increasing*the*attractiveness*of*the*novel* complex*call*relative*to*the*simple*call.*Here*we*addressed*the*specific*question*of* whether*an*appendage*could*restore*the*attractiveness*of*a*signal*with*inappropriate* intervals*(Schwartz*et*al.*2010)*or*pulses*of*suboptimal*duration*(Schul*&*Bush*2002).*

These*altered*signals*alone*were*less*attractive*than*standard*calls*with*speciesXtypical* pulse*periods*and*durations.*We*will*show*that*in*H.%versicolor*the*answer*is*positive*for* following*but*not*for*leading*appendages*over*a*wide*range*of*trill*and*appendage* durations.*

METHODS(

Animal'Collection'

In*2010X2012,*gravid*H.%versicolor%females*were*collected*from*local*breeding* ponds*in*Boone*County,*Missouri*(U.S.A.).*Females*were*placed*in*individual*containers* and*stored*in*an*iceXfilled*cooler*to*delay*oviposition.*All*females*were*returned*to*their* collection*site*after*experimentation,*usually*2X3*days*after*collection.*

Acoustic'Stimuli'

Synthetic*advertisement*calls*were*created*(16Xbits*per*sample,*20kHz*sampling* rate)*using*custom*software*(J.J.*Schwartz,*unpublished*software).*These*stimuli*were* modeled*after*natural*advertisement*call*and*have*been*found*to*be*as*attractive*as* prerecorded*natural*calls*(Gerhardt*2005b).*Each*stimulus*had*a*spectrum*consisting*of* 12* * * two*spectral*peaks*with*frequencies*(1.1*and*2.2*kHz,*relative*amplitude*of*the*1.1*kHz* peak*was*X6*dB)*close*to*the*mean*values*in*calls*of*male*H.%versicolor%from*the* populations*from*which*we*collected*females*(Gerhardt*2005a).*The*pulse*shape*in* synthetic*calls*was*also*similar*to*that*of*typical*natural*calls.**

We*generated*three*standard*calls*that*corresponded*to*short,*average,*and*long* calls*of*males*in*local*populations*(10X,*18X,*24Xpulses).*The*call*period*was*4*seconds*at*

20°*C*with*a*50%*pulse*duty*cycle*(25*ms*pulses*and*25*ms*silent*interval*between* pulses).*To*prevent*call*overlap*in*tests*using*longXduration*appendages,*the*call*period* was*increased*to*5*seconds.**

For*tests*using*incorrect*pulse*periods,*“gap”*calls*were*created*by*removing*one* pulse*from*a*standard*call,*resulting*in*one*abnormal*silent*interval*of*75*ms.%Gaps*were* created*by*dropping*pulses*from*one*of*three*possible*locations*in*the*call:*early,* midway,*or*late.*For*the*shortXcall*stimulus,*gaps*were*created*by*dropping*the*3rd,*5th,* or*9th*pulse*of*the*call;*for*the*average*stimulus,*dropped*pulses*were*either*the*5th,*9th,* or*14th*(Fig.*2X1);*and*the*longXcall*stimulus*had*either*the*5th,*12th,*or*20th*pulse* dropped.**

For*tests*using*pulses*of*suboptimal*duration,*affected*pulses*were*half*the* normal*pulse*duration*(12.5*ms)*with*rise/fall*time*ratios*(80%*of*pulse*is*rise*time,*20%* of*the*pulse*is*fall*time)*comparable*to*natural*pulses.*The*pulse*period*was*not* modified*to*maintain*a*50%*pulse*duty*cycle;*therefore*after*a*suboptimal*pulse*the* silent*interXpulse*interval*was*37.5*ms*instead*of*25*ms.*Suboptimal*pulses*(one*or*three*

13* * * pulses)*were*placed*starting*at*the*third*pulse*of*the*call*in*a*standard*call*containing* either*10,*18,*or*24*pulses.*

* All*appendages*were*created*by*the*tone*generator*function*in*Adobe*Audition* v2.0*(Adobe*Systems*Inc.,*San*Jose,*CA*USA)*and*had*the*same*spectrum*as*the*standard* and*altered*advertisement*calls.*Appendages*were*separated*from*the*advertisement* call*by*a*silent*interval*of*50*ms*(the*equivalent*of*one*pulse*period).*The*amplitudeX time*envelopes*of*the*appendages*and*the*silent*interval*with*respect*to*the* advertisementXcall*part*of*the*complex*calls*are*shown*in*Figures*2X3*through*2X8.*We* also*used*a*sound*level*meter*(described*in*Experimental*Procedure)*to*adjust*the* amplitude*of*appendages*independently*of*the*trilled*part*of*the*complex*call.*The*usual*

“fast”*RMS*value*of*the*standard*(220*ms)*appendage*was*91*dB*SPL*and*its*peak*value* was*within*about*0.5*dB*of*that*of*the*advertisementXcall*part*of*complex*calls*(see*also* the*bottom*oscillogram*of*Fig.*2X1).*In*experiments*using*a*longXduration*(657*ms)* appendage,*the*RMS*level*was*85*dB*SPL.*

Experimental'Procedure'

TwoXspeaker,*forcedXchoice*tests*were*conducted*in*a*temperatureXcontrolled* chamber*lined*with*wedges*of*anechoic*foam.*The*testing*chamber*and*equipment*used* in*these*experiments*have*been*described*previously*(Gerhardt*2005).*Females*were* acclimated*to*the*test*temperature*(20°*C*±*1.5°)*for*at*least*30*min*and,*after* adjustment*of*playback*levels,*were*placed*individually*in*a*release*cage*in*the*testing* chamber*for*one*minute*before*playbacks*began.*A*LarsenXDavis*800B*sound*level* meter*was*used*to*adjust*the*amplitude*of*synthetic*advertisement*calls*to*85*dB*SPL* 14* * * (sound*pressure*level*in*decibels*[dB]*re*20*μPa,*fast*RMS,*CXweighted)*at*the*point*of* the*release*cage;*this*value*is*within*the*natural*range*of*call*amplitude*at*1*m*(Gerhardt*

1975).**

After*three*repetitions*of*each*stimulus,*the*female*was*released*remotely*from* an*acoustically*transparent*cage*located*midway*between*two*loudspeakers*that*were*2* m*apart*and*facing*each*other.*Female*movements*and*locations*were*monitored* remotely*via*an*infraredXsensitive*camera*located*within*the*testing*chamber.*A* response*was*tabulated*when*a*female*exhibited*phonotaxis*(head*and*body*scans* correlated*with*the*occurrence*of*the*broadcast*of*a*synthetic*call)*and*moved*to*within*

10*cm*of*a*speaker*playing*a*stimulus.*Females*often*touched*the*wall*in*front*of*the* speaker.*

Females*were*tested*in*multiple*tests,*but*only*one*response*per*female*was* scored*in*any*one*test;*there*was*a*timeout*period*of*at*least*5*minutes*between* different*tests*of*the*same*female.*Females*that*did*not*reach*a*speaker*within*5* minutes*were*scored*as*“noXchoice”,*a*category*that*did*not*contribute*to*the*data*set.*

Some*of*these*females*were*retested*later.*All*experimental*procedures*were*approved* by*the*University*of*Missouri*Animal*Care*and*Use*Committee*(protocol*#1910).*

Data'presentation'and'statistical'estimates*

For*all*tests,*one*speaker*presented*the*standard*advertisement*call.*In*a*subset* of*tests,*the*second*speaker*played*an*altered*call*containing*either*a*“gap”*(a*dropped* pulse)*or*one*or*three*pulses*of*suboptimal*duration.*We*report*the*proportion*of* females*that*chose*the*altered*call.*In*another*series*of*tests,*the*standard* 15* * * advertisement*call*was*paired*with*an*alternative*of*an*altered*call*(“gap”*or*shortXpulse* call)*that*also*contained*an*appendage.*We*then*compared*the*proportion*of*females* choosing*the*alternative*call*containing*an*appendage*against*the*proportion*choosing* the*altered*call*alone*in*the*previous*test*against*the*standard*call.*

In*tests*comparing*the*effectiveness*of*calls*with*gaps*or*shortXduration*pulses,* we*show*the*95%Xcredible*intervals*around*the*proportion*that*chose*the*altered*call* rather*than*the*standard*call.*These*intervals*were*numerically*the*same*as*confidence* limits*because*we*assumed*a*uniform*prior*distribution;*they*are*interpreted*to*mean* that*the*true*proportion*had*a*95%*probability*of*occurring*within*the*limits.*We*also* report*the*results*of*twoXtailed*binomial*tests*of*the*null*hypothesis*that*the*standard* and*altered*calls*were*equally*attractive*(Table*1*in*supplementary*material).*Our*figures* show*the*differences*in*the*proportions*choosing*the*altered*call*and*the*proportions* choosing*the*altered*call*plus*an*appendage*as*described*above.*We*also*computed*2*X*

2*chiXsquare*tests*(or*Fisher’s*exact,*when*values*were*less*than*5)*using*the*numbers*of* females*that*chose*each*stimulus*in*the*two*tests.*The*results*of*these*tests*indicate* whether*or*not*the*restoration*was*statistically*significant*(Table*2X1).*

For*the*majority*of*our*tests*(66%),*the*females*used*in*the*standard*versus* altered*call*trial*were*different*than*females*used*in*trials*with*the*altered*call* containing*an*appendage.*The*remaining*tests*lacked*strict*independence*–*some* females*responded*in*both*tests*–*so*that*these*pXvalues*are*not*strictly*valid.*However,* previous*studies*with*North*American*treefrogs*(including*H.%versicolor)*have*shown*no* carryover*effects*in*repeated*trials*(Gerhardt*et*al.*2000).*In*any*event,*our*results*are* 16* * * better*interpreted*in*terms*of*the*overall*pattern*of*preferences*based*on*the*observed* proportions*and*credible*intervals*rather*than*on*the*significance*or*lack*thereof*of* individual*tests.*

RESULTS(

Effect'of'Inappropriate'Intervals'on'Call'Attractiveness'

Females*strongly*discriminated*against*calls*containing*gaps,*regardless*of*gap* location,*for*calls*of*short*or*average*duration.*For*stimuli*modeled*after*longXduration* calls,*females*significantly*discriminated*against*gaps*occurring*early*in*the*call,*but*this* discrimination*was*not*significant*for*gaps*occurring*midway*or*late*in*the*call*(twoX tailed*binomial*test,*p*<*0.05*for*significance*for*all*tests)*(Fig.*2X2).*

Effect'of'Appendage'Amplitude'and'Duration'on'Restoring'Relative'Attractiveness'

For*both*average*and*longXduration*advertisementXcall*types,*the*attractiveness* of*the*altered*advertisement*call*with*an*early*gap*was*enhanced*by*a*220*ms*following* appendage*with*an*RMS*amplitude*6*dB*greater*than*the*pulsed*portion*of*the*call*(85* dB*SPL)*(chiXsquare,*p*<*0.05)*(Fig.*2X3).*At*the*RMS*amplitude*of*85*dB,*appendage* duration*had*negligible*positive*effects,*especially*when*added*to*the*18Xpulse* advertisement*call*(Fig.*2X4).*In*a*direct*test,*females*significantly*preferred*the*shortX duration*(standard*220*ms)*appendage*over*the*longXduration*(657*ms)*appendage;* however*the*RMS*amplitude*of*the*short*appendage*was*6*dB*greater*than*that*of*the* long*appendage*(twoXtailed*binomial*test,*p*=*0.041).*These*results*were*the*basis*for* adjusting*the*amplitude*of*the*standard*appendage*(220*ms)*to*91*dB*RMS*

17* * * (approximately*equal*in*peak*amplitude*to*that*of*the*advertisement*call*part*of*the* complex*call)*in*all*of*the*other*tests*whose*results*are*presented*below.*

Effect'of'Gap'Location'on'Appendage'Effectiveness'

Appendages*were*most*effective*in*restoring*attractiveness*for*calls*that* contained*gaps*in*the*early*position,*and*significantly*more*females*preferred*this* appended*call*(chiXsquare,*p*<*0.05)*(Fig.*2X5).*While*proportionally*more*females*chose* appended*calls*with*a*gap*midway*or*late*in*the*pulse*train,*this*change*in*proportion* was*not*significant.*

Effect'of'Temporal'Order'

Leading*appendages*were*ineffective*at*enhancing*call*attractiveness.*In*fact,* females*significantly*discriminated*against*compound*calls*with*leading*appendages*

(twoXtailed*binomial*test,*p*<*0.05*for*all*tests,*n*=*20*for*all*tests).*The*change*in* proportion*of*females*choosing*the*leading*appendage*call*was*not*significantly* different*from*zero*(chiXsquare,*p*>*0.05)*(Fig.*2X6).*In*a*direct*test*between*leading*and* following*appendages,*females*had*marginally*significant*preference*for*appendages*in* the*following*position*(twoXtailed*binomial*test,*p*=*0.07,*n*=*31).**

Effects'of'Appendages'in'Restoring'Attractiveness'of'Calls'with'Suboptimal'Pulse'

Duration*

Females*significantly*discriminated*against*shortXduration*calls*with*one*or*three* pulses*of*suboptimal*duration*(Fig.*2X7;*twoXtailed*binomial*test,*p*<*0.05*for*all*tests,*n*

=*32).*When*the*call*was*of*average*duration,*however,*females*discriminated*only* against*calls*containing*three*suboptimal*pulses*(Fig*2X7;*twoXtailed*binomial*test,*n*=* 18* * * 30).*In*stimuli*modeled*after*longXduration*calls,*females*did*not*discriminate*calls* containing*one*or*three*suboptimal*pulses*(Fig.*2X7;*twoXtailed*binomial*test,*n*=*30*for* all*tests).**

When*a*following*appendage*was*added*to*a*shortXduration*call*with*suboptimal* pulses,*proportionally*more*females*chose*this*alternative*(Fig.*2X8;*chiXsquare,*p*<*

0.05).*The*addition*of*a*following*appendage*did*not*enhance*the*attractiveness*of*an* average*call*containing*one*suboptimal*pulse,*but*did*enhance*the*attractiveness*of*a* call*containing*three*suboptimal*pulses,*though*not*significantly*so*(Fig.*2X8;*chiXsquare,* p*=*0.104).*A*following*appendage*only*enhanced*a*longXduration*call*containing*one* suboptimal*pulse*and*did*not*enhance*a*call*containing*three*suboptimal*pulses*(Fig.*2X

8;*chiXsquare,*p*<*0.05*for*significance).*

Overall'Pattern'of'Restoration'by'Appendages'

Although*many*individual*tests*reached*statistical*significance,*we*argue*that*the* overall*pattern*of*female*preferences*is*more*important.*For*example,*our*results* include*22*pairs*of*stimuli*(altered*call*and*altered*call*with*a*following*appendage).*In*

19*out*of*22*of*these*tests*(86%)*there*was*a*10%*or*greater*increase*in*the*proportions* of*females*choosing*the*twoXelement*call*rather*than*the*standard*call*compared*with* the*proportion*of*females*choosing*the*altered*call*rather*than*the*standard*call.*

Furthermore,*we*found*that*in*16*out*of*22*tests*(73%),*the*restoration*was*statistically* significant*(p%<*0.05,*twoXtailed*binomial*tests).*In*these*tests*female*preference* changed*from*discrimination*against*the*altered*call*(tested*against*the*standard*call)*to*

19* * * a*lack*of*preference*for*the*standard*call*(13*of*22*tests)*or*even*a*preference*for*the* altered*call*plus*appendage*over*the*standard*call*(3*out*of*22*tests).*

DISCUSSION(

The*selectivity*of*females*in*our*behavioral*experiments*was*generally*consistent* with*that*of*temporally*selective*neurons.*Females*discriminated*against*calls*that* potentially*reset*counting*neurons*(gap*calls).*Our*experiments*also*showed*that*the* addition*of*an*acoustic*appendage*to*these*defective*calls*enhanced*call*attractiveness.*

Restoration*of*attractiveness*only*occurred,*however,*when*the*appendage*came*in*the* following*position*and*had*about*the*same*peakXtoXpeak*amplitude*as*the*pulsed* portion*of*the*call.*We*also*found*that*appendages*could*restore*attractiveness*of*calls* with*abnormally*shortXduration*pulses.*

Discrimination'Against'Calls'with'Gaps'

Females*significantly*discriminated*against*calls*that*contained*an*inappropriate* gap*(a*dropped*pulse).*This*result,*along*with*those*of*Schwartz*et*al.*(2010),*is* consistent*with*the*properties*of*intervalXcounting*neurons*reported*by*Rose*and* colleagues*(Edwards*et*al.*2002)*and*supports*their*prediction*that*intervalXcounting* neurons*would*influence*phonotactic*behaviour.*Although*intervalXcounting*neurons*are* described*as*being*sensitive*to*pulse*period,*females*of*H.%versicolor*are*selective*for* pulse*duration*and*interpulse*interval.*While*these*two*components*combine*to*result* in*a*specific*pulse*period*(and*its*reciprocal,*the*pulse*rate),*H.%versicolor*responds*to*a* wideXrange*of*pulse*periods*once*pulse*duration*equals*or*exceeds*a*minimum*value*

(Schul*&*Bush*2002).*We*found*the*same*phenomenon*in*our*present*tests*of*H.% 20* * * versicolor,*and*discrimination*against*calls*containing*pulses*of*suboptimal*duration*

(pulse*rate*is*unaltered)*was*especially*strong*in*calls*with*relatively*few*pulses.*The* results*of*neurophysiological*experiments*using*H.%versicolor%parallel*these*results,% indicating*that*the*intervalXcounting*neurons*in*H.%versicolor%also*require*that*the*pulses* have*the*correct*shape*(long*riseXtime)*as*well*as*interval*(G.*Rose*personal* communication).*The*effectiveness*of*slowXonset,*longXduration*pulses*in*driving* midbrain*auditory*neurons*may*explain*why*appendages*used*in*these*experiments* were*generally*attractive;*appendages*may*have*been*“equivalent”*to*a*single*pulse.*

Restoring'Call'Attractiveness'

Appendages*were*generally*effective*in*at*least*partially*restoring*the*call* attractiveness*for*trills*containing*inappropriate*gaps,*provided*that*the*appendage*had* the*same*peak*amplitude*as*the*trilled*part*of*the*complex*call.*Importantly,*call* restoration*or*enhancement*cannot*be*readily*explained*by*the*greater*duration*of* sensory*stimulation*provided*by*the*appendage.*This*idea*is*supported*by*the*fact*that* following*appendages*restored*or*enhanced*call*attractiveness*while*leading* appendages*did*not*(see*also*Gerhardt*et*al.*2007).*These*two*kinds*of*acoustic*stimuli* did*not*of*course*differ*in*duration*or*sound*energy.**

One*possible*mechanistic*explanation*for*these*behavioural*results*is*based*on* the*analysis*of*wholeXcell*patch*recordings*that*resolve*the*timing*of*excitation*and* inhibition*of*intervalXcounting*neurons*(G.*Rose,*personal*communication).*IntervalX counting*neurons*may*respond*strongest*at*the*onset*of*the*appendage,*so*that*for* appendages*in*the*leading*position,*a*long*gap*would*occur*between*this*excitation*and* 21* * * that*caused*by*the*second*element,*which*contains*pulses*and*intervals*that*most* effectively*drive*these*cells.*In*the*case*of*a*following*appendage,*the*gap*would*be* shorter*and*excitation*at*the*onset*of*the*following*appendage*might*result*in*temporal* summation*with*the*depolarization*caused*by*the*leading*pulse*train*(G.*Rose,*personal* communication).*Direct*tests*and*recordings*from*intervalXcounting*neurons*using*the* stimuli*from*this*study*are*necessary*to*confirm*this*explanation.*

Previous*studies*suggest*that*the*temporalXorder*effect*is*less*important*or*even* absent*in*the*closely*related*H.%chrysoscelis*(Seeba*et*al.*2010).*Thus*further*work* examining*the*effect*of*appendages*on*call*attractiveness*in*this*species*is*still*needed.*

Unpublished*results*indicate*that*many*of*the*counting*neurons*in*H.%chrysoscelis*are* selective*for*longXduration*pulses*as*well*as*fast*pulse*rates*(G.*Rose,*personal* communication).*A*tonal*appendage*in*the*leading*position*may*elicit*excitation*in* counting*neurons*and*would*be*just*as*effective*in*enhancing*the*attractiveness*of*calls* as*following*appendages.**

Additional*experiments*examining*the*neural*processes*that*influence*behaviour* are*important*for*a*more*complete*understanding*of*the*biology*of*behaviour,*and* without*this*type*of*integration,*interpretations*may*be*incomplete*or*inaccurate*(Ryan*

2005).*Collaboration*across*disciplines*often*suggests*important*experiments*that*may* not*be*intuitive*at*either*level.*Many*anuran*species*rely*on*the*temporal*properties* encoded*by*pulses*and*intervals*(Gerhardt*1982,*1988)*and*neurons*selective*for*pulse* period*have*been*found*in*multiple*species*(Edwards*et*al.*2007;*G.*Rose,*personal* communication).*The*selectivity*of*these*neurons*may*be*an*important*constraint*for* 22* * * call*evolution*for*species*with*pulsatile*call*structure.*More*research*is*needed*to* understand*how*common*sensory*mechanisms*may*affect*novel*call*attractiveness.*

*In*this*study,*behavioural*results*matched*predictions*from*the*neurophysiological* studies*regarding*the*importance*of*long*riseXtime*for*intervalXcounting*neurons*in*H.% versicolor.*Conversely,*behavioural*experiments*have*predicted*unique*properties*of*H.% versicolor*intervalXcounting*neurons,*such*as*the*effects*of*adding*pulses*of*suboptimal* duration.*The*response*properties*of*intervalXcounting*neurons*may*ultimately*offer*an* explanation*for*unanticipated*behavioural*results,*such*as*the*attractiveness*of*complex* calls*in*species*such*as*the*grey*treefrog.*Neither*males*of*this*species*nor*any*other* closely*related*species*produce*complex*calls.*

ACKNOWLEDGEMENTS(

We*thank*the*Gerhardt*lab,*past*and*present,*for*helpful*comments*and* assistance*with*frog*collection.*Katie*Youmans*assisted*with*female*choice*experiments.*

The*Social*Science*Statistics*Center*at*MU*provided*statistical*assistance.*We*also*thank*

Gary*Rose*for*feedback*on*this*manuscript.*Funding*was*provided*by*a*grantXinXaid*to*JJ*

Henderson*from*the*Society*for*Integrative*and*Comparative*Biology.*Frog*collection* followed*the*stipulations*of*a*scientific*collecting*permit*issued*by*the*Missouri*

Department*of*Conservation.*Experimental*procedures*approved*by*the*Institutional*

Animal*Care*and*Use*Committee*of*the*University*of*Missouri*(protocol*#1910).*

*

*

23* * * LITERATURE(CITED(

Adler,*T.*B.*&*Rose,*G.*J.*1998.*LongXterm*temporal*integration*in*the*anuran*auditory* system.*Nat%Neurosci,*1,*519–523.*

Adler,*T.*B.*&*Rose,*G.*J.*2000.*Integration*and*recovery*processes*contribute*to*the* temporal*selectivity*of*neurons*in*the*midbrain*of*the*northern*leopard*frog,*Rana* pipiens.*Journal%of%Comparative%Physiology%A:%Sensory,%Neural,%and%Behavioral% Physiology,*186,*923–937.*

Andersson,*M.*1994.*Sexual%Selection.%Princeton,*New*Jersey:*Princeton*University*Press.**

Arak,*A.*&*Enquist,*M.*1993.*Hidden*preferences*and*the*evolution*of*signals.* Philosophical%Transactions:%Biological%Sciences,*340,*207–213.*

Edwards,*C.*J.,*Alder,*T.*B.*&*Rose,*G.*J.*2002.*Auditory*midbrain*neurons*that*count.*Nat% Neurosci,*5,*934–936.*

Edwards,*C.*J.,*Leary,*C.*J.*&*Rose,*G.*J.*2007.*Counting*on*inhibition*and*rateXdependent* excitation*in*the*auditory*system.*Journal%of%Neuroscience,*27,*13384–13392.*

Gerhardt,*H.*C.*1975.*Sound*pressure*levels*and*radiation*patterns*of*the*vocalizations* of*some*North*American*frogs*and*toads.*Journal%of%comparative%physiology,*102,* 1–12.*

Gerhardt,*H.*C.*1982.*Sound*Pattern*Recognition*in*Some*North*American*Treefrogs* (Anura:*Hylidae):*Implications*for*Mate*Choice.*American%Zoologist,*22,*581–595.*

Gerhardt,*H.*C.*1988.*Acoustic*properties*used*in*call*recognition*by*frogs*and*toads.*In:* The%Evolution%of%the%Amphibian%Auditory%System,*(Ed.*by*B.*Fritzch,*M.*J.*Ryan,*W.* Wilczynski,*T.*E.*Hetherington,*&*W.*Walkowiak),*New*York:*Wiley.**

Gerhardt,*H.*C.*2005a.*Acoustic*spectral*preferences*in*two*cryptic*species*of*grey* treefrogs:*implications*for*mate*choice*and*sensory*mechanisms.*Anim%Behav,*70,* 39–48.*

Gerhardt,*H.*C.*2005b.*AdvertisementXcall*preferences*in*diploidXtetraploid*treefrogs* (Hyla*chrysoscelis*and*Hyla*versicolor):*implications*for*mate*choice*and*the* evolution*of*communication*systems.*Evolution,*59,*395–408.*

Gerhardt,*H.*C.*&*Huber,*F.*2002.*Acoustic%Communication%in%Insects%and%Anurans.% Chicago:*The*University*of*Chicago*Press.**

24* * * Gerhardt,*H.*C.*&*Schul,*J.*1999.*A*quantitative*analysis*of*behavioral*selectivity*for*pulse* riseXtime*in*the*gray*treefrog,*Hyla*versicolor.*Journal%of%Comparative%Physiology%A:% Sensory,%Neural,%and%Behavioral%Physiology,*185,*33–40.*

Gerhardt,*H.*C.,*Tanner,*S.*D.,*Corrigan,*C.*M.*&*Walton,*H.*C.*2000.*Female*preference* functions*based*on*call*duration*in*the*gray*tree*frog*(Hyla*versicolor).*Behavioral% Ecology,*11,*663–669.*

Gerhardt,*H.*C.,*Humfeld,*S.*C.*&*Marshall,*V.*T.*2007.*Temporal*order*and*the*evolution* of*complex*acoustic*signals.*Proceedings%of%the%Royal%Society%B:%Biological%Sciences,* 274,*1789–1794.*

Ryan,*M.*J.*2005.*The*evolution*of*behaviour,*and*integrating*it*towards*a*complete*and* correct*understanding*of*behavioural*biology.*Animal%Biology,*55,*419–439.*

Ryan,*M.*J.*&*Rand,*A.*S.*1993.*Sexual*selection*and*signal*evolution:*the*ghost*of*biases* past.*Philosophical%Transactions%of%the%Royal%Society%B:%Biological%Sciences,*340,* 187–195.*

Schul,*J.*&*Bush,*S.*L.*2002.*NonXparallel*coevolution*of*sender*and*receiver*in*the* acoustic*communication*system*of*treefrogs.*Proceedings%of%the%Royal%Society%B:% Biological%Sciences,*269,*1847–1852.*

Schwartz,*J.*J.,*Huth,*K.,*Hunce,*R.*&*Lentine,*B.*2010.*Effect*of*anomalous*pulse*timing* on*call*discrimination*by*females*of*the*gray*treefrog*(Hyla*versicolor):*behavioral* correlates*of*neurobiology.*J%Exp%Biol,*213,*2066–2072.*

Seeba,*F.,*Schwartz,*J.*J.*&*Bee,*M.*A.*2010.*Testing*an*auditory*illusion*in*frogs:* Perceptual*restoration*or*sensory*bias?*Anim%Behav,*79,*1317–1328.*

Tinbergen,*N.*1951.*The%study%of%instinct.%Clarendon*Press.**

Wilczynski,*W.*&*Endepols,*H.*2006.*Central*auditory*pathways*in*anuran*amphibians:* the*anatomical*basis*of*hearing*and*sound*communication.*In:*Hearing%and%Sound% Communication%in%Amphibians,*(Ed.*by*P.*M.*Narins,*A.*S.*Feng,*R.*R.*Fay,*&*A.*N.* Popper),*pp.*221–249.*Springer.*

25* * * !

!

Figure(2:1.(Examples*of*stimuli*used.*Oscillograms*illustrating*gap*location*in*a*standard* call*duration*(18*pulses)*with*the*average*number*(18)*of*pulses.*Gaps*were*the* equivalent*of*one*dropped*pulse,*resulting*in*a*75*ms*interval.( *

26* * * ! 27* * * Figure(2:2.*Discrimination*against*calls*with*gaps*(one*missing*pulse).*Females*preferred* normal*advertisement*calls*over*“gap”*calls*containing*one*dropped*pulse*for*calls* modeled*after*(a)*short,*(b)*average,*and*(c)*long*duration*calls.*Dashed*line*indicates* proportion*choosing*alternative*based*on*chance*(50%).*Bars*are*95%*credible*intervals.* **denotes*p*<*0.05*(twoXtailed*binomial*test).*(

28* * * ! Figure(2:3.(Effect*of*relative*intensity*of*the*appendage.*Difference*in*the*proportion*of* females*choosing*a*call*with*a*missing*pulse*plus*an*appendage*as*a*function*of*the* amplitude*of*an*appendage*of*220*ms.*The*standardXcall*alternative*to*calls*with*a* missing*pulse*(one*with*and*one*without*the*appendage)*had*18*pulses.*Bars*show*the* upper*95%*credible*intervals.***denotes*p*<*0.05*(Fisher’s*exact*or*chiXsquare,*df*=*1).( !

!

!

!

!

!

29* * * !

Figure(2:4.(Effect*of*appendage*duration.*Difference*in*the*proportion*of*females* choosing*a*call*with*a*missing*pulse*plus*an*appendage*of*equal*amplitude*and* proportion*choosing*the*standard*call*to*the*altered*call*alone.*Neither*the*220Xms*nor* the*657Xms*appendages*were*effective*in*restoring*attractiveness*to*the*altered* advertisement*call.*Both*appendages*were*especially*ineffective*in*combination*with*the* altered*call*with*18*pulses.*■*=*10*pulse*call;*▲=*18*pulse*call;*□*=*24*pulse*call.*n*=*20* for*all*tests.*Bars*are*the*upper*95%*credible*intervals.***denotes*p*<*0.05.*(Fisher’s*exact* or*chiXsquare,*df*=*1).( !

!

!

!

!

30* * * !

Figure(2:5.(Effect*of*gap*location.*Difference*in*proportion*of*females*choosing*a*call* with*a*missing*pulse*plus*an*appendage*and*the*proportions*choosing*between*the* standard*call*and*the*altered*call*alone.*“Gap”*calls*with*a*dropped*pulse*early*in*the* pulse*train*were*enhanced*the*most*when*an*appendage*(220*ms;*91*dB*SPL,*fast*rms)* was*added.*This*figure*shows*the*results*for*tests*of*calls*of*average*call*duration*(18* pulses);*bars*are*the*upper*95%*credible*intervals.***denotes*p*<*0.05.*(Fisher’s*exact*or* chiXsquare,*df*=*1).( !

!

!

!

!

!

31* * * !

Figure(2:6.(Effect*of*temporal*order.*Females*preferred*appendages*(220*ms;*91*dB*SPL,* fast*rms)*in*the*following*position*but*discriminated*against*calls*with*the*same* appendage*in*the*leading*position.*Results*for*testing*using*average*call*duration*(18* pulses)*are*shown.*Bars*are*upper*95%*credible*intervals.***denotes*p*<*0.05.*(Fisher’s* exact*or*chiXsquare,*df*=*1).( !

! ! ! ! ! ! ! ! ! ! ! ! ! !

32* * * ! Figure(2:7.*Discrimination*against*calls*with*one*or*three*abnormally*short*pulses.* Shown*are*the*proportions*choosing*such*calls*rather*than*a*standard*call*with*the*same* number*of*equalXduration*pulses.*In*most*tests,*females*discriminated*against*calls* containing*suboptimalXduration*pulses*(with*some*exceptions),*especially*if*it*contained* three*of*these*pulses.*■*=*10*pulse*call;*▲=*18*pulse*call;*□*=*24*pulse*call.*Dashed*line* indicates*proportion*choosing*alternative*based*on*chance*(50%).*Bars*are*upper*95%* credible*intervals.***denotes*p*<*0.05*(twoXtailed*binomial*test).( ! ! ! ! ! ! ! !

33* * * ! Figure(2:8.(Effect*of*appendages*in*restoring*attractiveness*of*call*with*suboptimal* pulses.*Difference*in*the*proportion*of*females*choosing*a*call*with*pulses*of*subX optimal*duration*plus*an*appendage*and*the*proportion*choosing*the*standard*call*to* the*altered*call*alone.*More*females*chose*the*call*with*suboptimal*duration*pulses*if*it* included*a*following*appendage*(220*ms;*91*dB*SPL).*This*enhancement*of* attractiveness*had*the*most*effect*on*calls*of*short*duration.*■*=*10*pulse*call;*▲=*18* pulse*call;*□*=*24*pulse*call.*Bars*show*the*upper*95%*credible*intervals.***denotes*p*<* 0.05.*(Fisher’s*exact*or*chiXsquare,*df*=*1).(

34* * * TABLE(1:1:(Choices*of*females*in*individual*behavioral*tests.( ! Test...... Choosing.STD...... Choosing.ALT. ...Binomial.pF value. 10pul!Early!Gap! 32! 0! <0.0001! 10pul!Mid!Gap! 30! 2! <0.0001! 10pul!Late!Gap! 24! 6! 0.0014! 18pul!Early!Gap! 16! 4! 0.0118! 18pul!Mid!Gap! 22! 8! 0.0161! 18pul!Late!Gap! 22! 8! 0.0161! 24pul!Early!Gap! 15! 5! 0.0414! 24pul!Mid!Gap! 17! 13! 0.5847! 24pul!Late!Gap! 20! 10! 0.0987! !

Test...... Choosing.STD...... Choosing.ALT. ..ChiFsquare.pF value. 18pul!Gap! 16! 4! <

Test...... Choosing.STD...... Choosing.ALT. ..ChiFsquare.pF value. 10pul!Gap! 30! 2! <

35* * * Test...... Choosing.STD...... Choosing.ALT. ..ChiFsquare.pF value. 10pul!Mid!Gap! 30! 2! <

Test...... Choosing.STD...... Choosing.ALT. ..ChiFsquare.pF 36* * * value. 10pul!Subpulse! 22! 10! <

37* * * CHAPTER.3.

EFFECT.OF.TEMPORAL.PROPERTIES.ON.ATTRACTIVENESS.OF. NOVEL.COMPLEX.CALLS.IN.THE.COPE’S.GREY.TREEFROG,.HYLA% CHRYSOSCELIS%.

Jennifer!J.!Henderson!and!H.!Carl!Gerhardt!

Division!of!Biological!Sciences,!University!of!Missouri,!Columbia,!MO!65211

38* * * INTRODUCTION(

For*many*animals,*acoustic*signals*are*important*for*aggressive*interactions*

(Brown*et%al.,*2006;*Reichert*&*Gerhardt,*2014),*individual*recognition*(McComb*et%al.,* and*choosing*suitable*mates*(Andersson,*1994;*Gerhardt*&*Huber,*2002).*The*role*of* acoustic*signals*in*mate*choice*behavior*has*been*widely*studied*from*both*the* perspective*of*signal*production*and*the*receiver’s*sensory*system.*While*some*signals* may*contain*information*about*the*quality*of*a*potential*mate*(Johnstone,*1995;*Cotton* et%al.*2004),*signals*may*also*exploit*preXexisting*sensory*biases*or*hidden*preferences*

(Arak*&*Enquist,*1993;*Endler*&*Basolo,*1998;*Ryan*&*Rand,*1993).*

In*frogs,*the*addition*of*acoustic*elements*or*appendages*to*simple*signals*may* exploit*sensory*biases*and*enhance*the*attractiveness*to*female*receivers*(Ryan*&*Rand,*

1993).*Furthermore,*experiments*have*demonstrated*that*the*addition*of*an*entirely* novel*acoustic*appendage*(such*as*a*tone*or*burst*of*noise)*can*enhance*call* attractiveness,*even*in*species*that*only*produce*simple*calls*(Gerhardt*et%al.,*2007;*

Henderson*&*Gerhardt,*2013;*Ryan*et%al.,*2010;*Seeba*et%al.,*2010).*

We*examined*the*effect*of*novel*acoustic*appendages*on*call*attractiveness*in* the*Cope’s*gray*treefrog,*Hyla%chrysoscelis.*This*species,*along*with*the*closely*related*H.% versicolor,*is*an*excellent*model*for*studying*novel*complexity*as*males*only*produce* simple,*trilled*advertisement*calls*but*females*respond*to*novel*complex*calls*in*the* laboratory.*Although*closely*related*(H.%chrysoscelis,*is*one*of*the*diploid*progenitors*of* the*tetraploid*H.%versicolor*(Holloway*et%al.,*2006;*Ptacek*et%al.,*1994),*the*two*species* use*different*fineXtemporal*properties*for*call*recognition.*H.%chrysoscelis*females*are*

39* * * highly*selective*for*pulse*rate,*while*H.%versicolor*females*are*more*tolerant*of*a*range* of*pulse*rates*and*instead*evaluate*pulse*duration,*pulse*shape,*and*the*silent*interval* between*pulses*(Schul*&*Bush,*2002).*Both*species*use*grossXtemporal*properties,*such* as*the*number*of*pulses*per*call*and*callXrepetition*rate*to*choose*among*conspecific* males.*

** Previous*experiments*illustrated*that*temporal*order*of*the*novel*appendage* and*the*established*call*of*the*species*was*an*important*factor*in*the*attractiveness*of* complex*calls*(Gerhardt*et%al.,*2007).*This*result*may*be*explained*by*a*class*of* temporally*selective*(intervalXcounting)*neurons*that*were*recorded*in*H.%regilla*and*

Rana%pipiens.*These*cells*were*sensitive*to*pulse*period*(the*beginning*of*one*pulse*to* the*beginning*of*the*next)*and*fired*after*the*presentation*of*a*threshold*number*of* correct*pulse*periods*at*a*point*when*enhanced*excitation*overcame*inhibition*(Adler*&*

Rose,*1998;*Edwards*et%al.,*2002).*Incorrect*pulse*periods*(too*long*or*too*short)*reset* the*counting*process*(Edwards*et*al.,*2002;*Edwards*et%al.,*2007).*Once*these*neurons* responded,*the*majority*continue*to*fire*tonically*for*the*entire*duration*of*the*stimulus*

(Adler*&*Rose,*2000).*IntervalXcounting*neurons*(ICNs)*have*been*identified*in*multiple* anuran*species*and*are*located*in*the*torus*semicircularis*(a*homologue*of*the*inferior* colliculus*of*higher*vertebrates).*This*area*integrates*forebrain*inputs*and*interfaces* with*the*motor*system*(Wilczynski*&*Endepols,*2006)*and*thus*is*likely*important*for* phonotaxis*(the*movement*towards*sound).*

* Schwartz*et*al.*(2010)*and*Henderson*&*Gerhardt*(2013)*found*that*the* phonotactic*selectivity*of*female*H.%versicolor%was*consistent*with*the*response*

40* * * properties*of*temporallyXselective*neurons.*Calls*with*inappropriate*pulse*periods*

(dropped*pulses)*or*suboptimal*duration*were*discriminated*against*in*female*choice* experiments.*Furthermore,*call*attractiveness*was*restored*to*some*extent*if*the*altered* call*contained*specific*following*appendages*(Henderson*&*Gerhardt,*2013).*One* explanation*for*why*following*appendages*were*effective*in*“rescuing”*unattractive*calls* may*be*the*tonic*mode*of*firing*of*the*intervalXcounting*neurons.*After*a*threshold* number*of*correct*pulse*periods,*the*neurons*may*continue*to*respond*during*an* appendage*that*followed*the*pulses.*Appendages*that*lead*the*pulsed*portion*of*the*call* might*not*effectively*stimulate*the*onset*of*or*even*inhibit*the*intervalXcounting* neurons.*

Our*main*goal*in*this*experiment*was*to*examine*the*effects*of*novel*acoustic* appendages*on*call*attractiveness*in*H.%chrysoscelis.*Because*female*frogs*and*intervalX counting*neurons*are*selective*for*the*same*call*parameters,*we*expected*inappropriate* gaps*in*the*calls*to*be*unattractive.*Previous*experiments*with*H.%chrysoscelis*have* shown*that*females*often*respond*to*appendages*in*the*leading*as*well*as*following* position*(Gerhardt*et%al.,*2007;*Seeba*et*al.,*2010).*This*was*not*particularly*surprising* because*of*the*differences*already*noted*between*this*species*and*H.versicolor.*Here*we* tested*H.%chrysoscelis*in*experiments*that*mirrored*a*previous*study*using*H.%versicolor%

(Henderson*&*Gerhardt,*2013).%In*addition*to*determining*whether*appendages*could* restore*call*attractiveness*of*signals*containing*inappropriate*pulse*periods*regardless*of* the*temporal*order,*we*investigated*the*effects*of*appendage*amplitude*and*the*

41* * * duration*of*the*silent*interval*between*the*appendage*and*the*advertisementXcall* element.*

METHODS(

Animal'collection'

During*the*summers*of*2010X2013,*we*collected*gravid*H.%chrysoscelis*females* from*breeding*ponds*located*in*Phelps*County,*Missouri*(U.S.A.).*Females*were*placed* in*individual*containers*in*an*iceXfilled*cooler*to*delay*oviposition.*One*hundred*of*105*

(95%)*females*responded*in*one*or*more*tests*(mean*of*4.2*tests/female).*After* behavioral*experiments,*all*frogs*were*returned*to*their*point*of*collection*within*a* week.*

Acoustic'stimuli'

* Using*custom*software*(J.J.*Schwartz,*unpublished*software),*we*created* synthetic*advertisement*calls*(16Xbits*per*sample,*20kHz*sampling*rate)*that*modeled* the*natural*advertisement*call*of*the*species.*Calls*had*two*spectral*peaks*(1.2*and*2.4* kHz,*relative*amplitude*of*the*1.2*kHz*peak*was*X6*dB)*and*had*pulse*shape*similar*to* that*of*natural*calls.*Previous*studies*found*that*such*signals*were*as*attractive*as* typical,*preXrecorded*calls*(Gerhardt,*2005b).*

* In*each*test,*females*were*presented*with*a*standard*call*that*corresponded*to* an*average*duration*call*(36Xpulses)*of*males*from*the*population*in*Phelps*County,*MO*

(37°*37'*6.41"*N,*91°*59'*54.02”*W)*from*which*females*were*tested*(Fig.*3X1).*Females* were*also*presented*with*an*alternative*call,*which*we*describe*below.*The*call*period*of*

42* * * each*stimulus*was*4*seconds*at*20°*C*and*each*call*had*a*50%*pulse*duty*cycle*(10*ms* pulses*and*10*ms*silent*intervals).*

* The*alternative*calls*used*in*this*experiment*contained*an*abnormally*long*silent* interval*by*the*removal*of*one*or*two*pulses,*hereafter*termed*a*“gap*call”.*The* dropped*pulse*was*always*the*7th*(and*8th*pulse*for*two*dropped*pulses)*of*the*call.*This* results*in*a*silent*interval*of*30*ms*for*one*dropped*pulse*and*50*ms*for*two*dropped* pulses*(Fig.*3X1).*Both*the*standard*call*and*the*pulsed*portion*of*the*alternative*calls* were*presented*with*amplitude*of*85*dB*SPL*at*1*m*(sound*pressure*level*in*decibels*

[dB]*re*20*μPa,*fast*RMS,*CXweighted*using*a*LarsenXDavis*800B*sound*level*meter).*

* Most*alternative*calls*included*a*tonal*appendage.*These*appendages*were* created*using*the*tone*generator*function*in*Adobe*Audition*v2.0*(Adobe*Systems*Inc.,*

San*Jose,*CA*USA).*Appendages*had*the*same*spectral*properties*as*the*pulses*(1.2*and*

2.4*kHz*frequencies)*and*had*25*ms*riseX*and*fallXtimes.*Tones*were*separated*from*the* pulsed*portion*of*the*call*by*a*silent*interval*of*either*20*ms*or*50*ms.*The*value*of*20* ms*is*equivalent*to*one*pulse*period*for*this*species*(however,*the*period*between*the* last*pulse*and*the*appendage*is*30*ms)*and*the*value*of*50*ms*was*selected*to*mirror*an* experiment*using*H.%versicolor%(Henderson*&*Gerhardt,*2013).*Our*experiment*used* three*variants*of*this*tonal*appendage:*standard,*loud,*and*long.*The*standard* appendage*was*160*ms*in*duration*and*the*fast*RMS*value*was*85*dB*SPL.*The*loud* appendage*had*the*same*duration*as*the*standard,*however*its*amplitude*was* increased*by*6*dB*(91*dB*SPL*fast*RMS).*The*long*appendage*variant*was*485*ms*in* duration*and*had*amplitude*of*85*dB*SPL*fast*RMS*(Fig.*3X1).*

43* * * Experimental'procedure*

* All*tests*were*performed*in*the*temperatureXregulated,*semiXanechoic*testing* chamber*that*has*been*previously*described*(Gerhardt,*2005a,*2005b).*Females*were* acclimated*to*the*test*temperature*(20°*C*±*1.5°)*in*an*incubator*for*30X40*min.*At*the* start*of*each*experimental*trial,*females*were*placed*in*a*small,*acoustically*transparent*

(made*of*hardware*cloth)*release*cage*located*midway*between*two*speakers*(distance* between*speakers*is*2*m).*An*SPL*meter*was*used*to*adjust*the*amplitude*of*the* standard*advertisement*call*to*85*dB*SPL*fast*RMS*at*the*point*of*the*release*cage.*

* Females*were*kept*in*the*release*cage*for*one*minute*before*playbacks*began.*

After*three*repetitions*of*each*stimulus,*females*were*released*remotely*and*the* movements*of*each*frog*were*monitored*using*an*infraredXsensitive*camera.*All*trials* were*twoXspeaker,*forcedXchoice*tests*in*which*a*response*was*counted*if*a*female* exhibited*phonotaxis*(orientation*correlated*with*the*broadcast*of*a*call)*and*touched* the*arena*wall*directly*in*front*of*a*speaker.*Only*one*response*per*female*was*used*in* any*one*test,*but*many*females*were*used*in*multiple*tests.*To*reduce*the*chance*of* carryXover*effects,*which*however*have*not*been*detected*in*this*species*using*the* protocol*just*described*(Gerhardt*et%al.,*2000),*there*was*a*timeout*period*of*at*least*5* minutes*between*different*tests*using*the*same*female.*If*a*female*did*not*reach*a* speaker*within*5*minutes,*it*was*scored*as*“noXchoice”*and*did*not*contribute*to*the* data*set.*All*experimental*procedures*were*approved*by*the*University*of*Missouri*

Animal*Care*and*Use*Committee*(protocol*#1910).*

Statistical'analysis'

44* * * For*the*majority*of*tests,*the*standard*advertisement*call*was*presented*from* one*speaker.*The*second*speaker*played*the*alternative*call*that*contained*a*gap*

(dropped*pulses)*and*in*a*subset*of*tests,*the*alternative*gap*call*included*a*tonal* appendage.*We*report*the*proportion*of*females*that*chose*each*alternative*and*show*

95%*credible*intervals,*which*are*numerically*the*same*as*confidence*limits*because*we* assumed*a*uniform*prior*distribution.*We*also*report*the*results*of*twoXtailed*binomial* tests*of*the*null*hypothesis*that*alternatives*were*equally*attractive.*

RESULTS(

Experiment'1:'Two'gaps'in'the'call'are'required'to'reduce'call'attractiveness'

When*an*advertisement*call*contained*one*dropped*pulse*(creating*a*30*ms* gap),*females*did*not*significantly*discriminate*against*this*call*type*compared*to*the* standard*advertisement*call*(twoXtailed*binomial*test,*P*=*0.503,*N*=*20)*(Fig.*3X2).*

However,*when*the*call*contained*two*dropped*pulses*(creating*a*50*ms*gap),*females* significantly*discriminated*against*this*gap*call*compared*to*the*standard*(twoXtailed* binomial*test,*P*<*0.05,*N*=*20)*(Fig.*3X2).*For*this*reason,*for*all*subsequent*tests,*gap* calls*had*two*dropped*pulses.*

Experiment'2:'Appendage'amplitude'and'restoration'of'call'attractiveness'

We*first*presented*females*with*a*choice*between*the*standard*advertisement* call*and*a*gap*call*that*also*included*one*acoustic*appendage.*All*appendages*in*this*and* the*following*experiment*were*separated*from*the*pulse*train*by*50*ms.*This*allowed*us* to*compare*our*results*directly*with*a*previous*study*using*the*closely*related*species*H.% versicolor*(Henderson*&*Gerhardt,*2013).*Females*significantly*discriminated*against*

45* * * gap*calls*with*appendages*in*the*following*position*(after*the*pulses)*for*both*standard*

(160*ms*duration,*85*dB*SPL)*and*long*(485*ms*duration,*85*dB*SPL)*appendages*(twoX tailed*binomial*test,*P*<*0.05**and*N*=*20*for*both*tests)*(Fig.*3X3).*When*amplitude*of* the*appendage*was*increased*by*6*dB*(160*ms*duration,*91*dB*SPL),*females*no*longer* discriminated*against*the*gap*call*type*compared*to*the*standard*advertisement*call*

(twoXtailed*binomial*test,*P*=*0.263,*N*=*20)*(Fig.*3X3).**

Experiment'3:'Temporal'order'of'call'and'appendage:'50'ms'silent'interval'

In*experiment*2,*we*found*that*attractiveness*of*a*gap*call*was*restored*with*a* loud,*following*appendage*(160*ms*duration,*91*dB*SPL)*(Fig.*3X4).*For*experiment*3,*we* presented*females*with*the*same*call*components*but*with*the*appendage*in*the* leading*position*(the*pulsed*portion*of*the*call*followed*the*appendage*by*50*ms).*

Leading*appendages*did*not*restore*call*attractiveness*as*females*significantly* discriminated*against*the*alternative*call*containing*a*leading*appendage*(twoXtailed* binomial*test,*P*=*0.012,*N*=*20)*(Fig.*3X4).*When*we*presented*females*with*a*direct*test* of*gap*calls*with*a*following*appendage*versus*one*containing*a*leading*appendage,* there*was*no*significant*difference*in*preference*(twoXtailed*binomial*test,*P*=*0.824,*N*

=*20)*(Fig.*3X5).*

Experiment'4:'Temporal'order'of'call'and'appendage:'20'ms'silent'interval'

A*loud*appendage*that*followed*the*pulsed*portion*of*the*call*by*50*ms*restored* call*attractiveness,*however*this*effect*was*lost*when*the*silent*interval*was*20*ms*(twoX tailed*binomial*test,*P*<*0.05,*N*=*20)*(Fig.*3X6a).*Conversely,*while*a*leading*appendage* separated*by*the*pulsed*portion*by*50*ms*did*not*restore*attractiveness,*females*no*

46* * * longer*discriminated*against*the*alternative*with*the*leading*appendage*call*when*the* gap*between*the*elements*was*20*ms*(twoXtailed*binomial*test,*P*=*0.503,*N*=*20)*(Fig.*

3X6b).*

DISCUSSION(

In*this*experiment*we*found*that*novel*acoustic*appendages*were*capable*of* restoring*call*attractiveness*in*H.%chrysoscelis.*As*in*the*results*of*a*study*of*the*closely* related*H.%versicolor,*females*did*not*discriminate*against*gap*calls*when*the*alternative* included*a*relatively*loud*appendage*in*the*following*position*with*a*silent*interval*of*50* ms.*The*length*of*the*silent*interval*between*the*pulsed*portion*of*the*call*and*the* appendage*affected*the*relative*attractiveness*of*the*compound*call.*

Discrimination'Against'“Gap”'Calls'

As*in*H.%versicolor,%females*of*H.%chrysoscelis%discriminated*against*calls* containing*abnormally*long*gaps*in*the*pulse*train*(Henderson*&*Gerhardt,*2013;*

Schwartz*et%al.,*2010).*These*behavioral*results*are*consistent*with*expectations*based* on*the*response*properties*of*intervalXcounting*neurons*reported*by*Edwards*et%al.*

(2002).*Unlike*H.%versicolor,*however,*we*found*that*H.%chrysoscelis%females*only* discriminated*against*calls*that*contained*a*gap*equivalent*to*two*dropped*pulses*(50* ms*gap;*Fig.*3X2).*Resetting*of*intervalXcounting*neurons*typically*occurs*when* inappropriate*gaps*are*at*least*two*pulse*periods*in*length*(G.*Rose,*personal* communication).*The*silent*interval*caused*by*the*removal*of*one*pulse*thus*might*be* inadequate*to*reset*temporally*selective*neurons.*

Effectiveness'of'Calls'with'Tonal'Appendages:'Temporal'Order'

47* * * Both*following*and*leading*appendages*sometimes*restored*the*attractiveness*of* otherwise*unattractive*gap*calls.*When*the*silent*interval*between*the*pulsed* advertisement*call*element*and*the*appendage*was*50*ms,*our*results*were*similar*to* comparable*experiments*with*H.%versicolor.*Compound*calls*with*appendages*that* followed*the*pulse*train*were*often*more*attractive*than*the*advertisement*call*alone*

(Gerhardt*et%al.,*2007)*or*more*effective*in*rescuing*call*attractiveness*(Henderson*&*

Gerhardt,*2013)*than*were*appendages*that*led*the*advertisement*call*element*(Fig.*3X

2).*This*order*preference*was*nevertheless*weaker*in*H.%chrysoscelis*than*in*H.%versicolor* in*that*there*was*no*preference*in*a*direct*test*for*a*compound*call*with*a*following* appendage*over*a*compound*call*with*a*leading*appendage*(Fig.*3X3).*'

* Seeba*et*al.*(2010)*found*that*advertisement*calls*of*H.%chrysoscelis*with*a*three* noiseXburst*appendage*(each*noise*burst*had*a*duration*of*99*ms)*separated*by*a*silent* interval*of*11*ms*were*more*attractive*than*the*advertisement*call*alone*in*the*leading* position*but*not*statistically*so*in*the*following*position.*They*also*found*no*difference* in*direct*tests*of*the*attractiveness*of*advertisement*calls*with*noiseXburst*appendages* in*the*leading*and*following*appendages.*Gerhardt*et*al.*(2007)*also*noted*that* compound*calls*with*leading*appendages*were*somewhat*more*likely*to*be*preferred*to* the*advertisement*call*alone*in*H.%chrysoscelis*than*in*H.%versicolor.*While*these*two* experiments,*as*well*as*the*results*from*this*study,*are*not*completely*comparable,*the* behavioral*differences*observed*between*populations*of*H.%chrysoscelis*is*intriguing.*

When*viewed*as*a*whole,*the*three*noiseXburst*appendages*used*by*Seeba*et%al.*(2010)* had*much*longer*duration*than*those*used*by*Gerhardt*et%al.*(2007).*However,*based*on*

48* * * the*results*from*this*study*(Figure*3X3)*appendage*duration*alone*does*not*explain*this* effect.*The*difference*in*silent*interval*between*appendages*and*pulses*(11*ms*in*Seeba* et%al.*(2010);*50*ms*in*Gerhardt*et%al.,*2007))*may*explain*the*observed*preference,*or* lack*thereof,*for*leading*appendages.*

Effect'of'the'Silent'Interval'between'Appendages'and'Pulses'in'Leading'Appendages'

The*results*discussed*in*the*previous*paragraph*were*cited*as*reasons*for* doubting*that*the*phenomenon*of*forward*masking*contributed*in*any*significant*way*to* explaining*the*results*of*tests*in*which*calls*with*leading*appendages*were*less* attractive*than*the*advertisement*call*alone*(Gerhardt*et%al.,*2007).*We*emphasize,* however,*that*in*all*three*of*these*studies,*the*silent*interval*between*the*elements*of* compound*calls*was*fixed.*Perhaps*the*most*significant,*and*certainly*the*most*puzzling,* results*of*our*new*study*concern*the*effects*of*changing*this*silent*interval.*Reducing* the*silent*interval*to*20*ms*significantly*increased*the*effectiveness*of*a*compound*call* with*a*leading*appendage*in*restoring*the*attractiveness*of*a*gap*call*while*significantly* reducing*the*effectiveness*of*a*compound*call*with*a*following*appendage*(Figure*3X6).*

Preliminary*results*of*experiments*in*which*the*silent*interval*was*varied* systematically*are*consistent*in*that*leading*tonal*appendages*separated*by*the*pulsed* portion*of*the*call*by*a*silent*interval*of*10*and*40*ms*were*more*effective*in*gapXcall* restoration*than*a*silent*interval*of*100*ms*(as*well*as*the*50Xms*interval).*Similarly,* following*appendages*were*less*effective*at*a*silent*interval*of*10*ms*and*more*effective* at*a*silent*interval*of*100*ms.*Studies*of*the*effects*of*varying*the*silent*interval*in* compound*calls*over*a*greater*range*on*the*preferences*of*H.%versicolor*also*show*how*

49* * * this*variable*interacts*with*other*properties*to*affect*relative*attractiveness*of* compound*and*simple*calls*(Gerhardt*&*Humfeld,*in*preparation).**

Species'Differences'

In*the*Introduction*we*pointed*out*some*profound*differences*in*the*preference* criteria*of*the*two*cryptic*species*of*gray*treefrog*with*regard*to*fineXscale*temporal* properties*of*their*advertisement*call.*Not*surprisingly,*our*present*paper*illustrates*that*

H.%chrysoscelis%differs*from*H.%versicolor*in*terms*of*how*appendages*affect*the* restoration*of*gap*calls*(Henderson*&*Gerhardt,*2013).*The*neural*mechanisms*behind* these*behavioral*differences*are*likely*to*involve*temporally*selective*neurons.*Rose*and* his*colleagues*have*already*discovered*both*quantitative*and*qualitative*differences* between*the*species*in*responses*to*the*same*auditory*stimuli*(G.*Rose,*personal* communication).*Some*cells*that*are*selective*for*fast*pulse*periods,*for*example,*often* show*strong*inhibition*directly*after*the*end*of*a*series*of*pulses*(Edwards*et%al.,*2007;*

Rose*et%al.,*2011);*additional*pulses*at*the*optimal*periods*may*reverse*this*inhibition.*

For*following*appendages,*however,*longer*offsets*between*the*pulse*train*and*a*tonal* appendage*may*permit*the*relatively*weak*excitatory*effects*of*the*latter*to*combine* with*rebound*from*inhibition*that*follows*the*former.*Auditory*neurons*that*respond*to* slow*pulseXrate*stimuli*have*also*been*identified*(G.*Rose,*personal*communication).*For* these*neurons,*longXduration*pulses*and*perhaps*tonal*appendages*appear*to*elicit*a* train*of*neuronal*spikes*in*the*excitatory*afferents*that*mimic*a*train*elicited*by*a*series* of*pulses.*Stimulation*of*such*cells*by*leading*tonal*appendages*may*underlie*the* restoration*of*call*attractiveness*observed*in*our*behavioral*experiments.*Additional*

50* * * neurophysiological*experiments*in*which*recordings*from*both*classes*of*ICNs*are*tested* with*stimuli*modeled*after*those*we*used*in*our*behavioral*experiments*(in*which*both* the*silent*portion*length*and*temporal*order*were*varied)*are*necessary*and*may* provide*a*test*of*this*hypothesis.*

Studies*examining*behavior*and*underlying*mechanisms*in*multiple*species*are* important*for*understanding*possible*sensory*biases.*The*presence*of*temporally* selective*neurons*that*respond*to*longXduration,*slow*pulse*rate*stimuli*in*H.% chrysoscelis,*but*not*in*the*closely*related*H.%versicolor,*could*have*interesting* implications*for*signal*preference*and*evolution.*While*adding*additional*pulses*is*most* effective,*novel*acoustic*appendages*are*still*successful*in*enhancing*call*attractiveness*

(Seeba*et*al.,*2010).*Finally,*preferences*based*on*call*duration*(number*of*pulses*in*a* call)*are*nonXlinear*in*both*species*(Bee,*2008;*Gerhardt*et*al.,*2000).*Given*the*same* percentage*difference*in*duration,*preferences*for*the*longer*of*two*calls*were*stronger* when*the*absolute*durations*of*the*alternatives*were*short*rather*than*long.*This*means* that*the*absolute*durations*of*both*advertisement*call*and*appendages*are*likely*to*be* relevant*properties*that*should*be*considered*in*future*behavioral*and* neurophysiological*studies.*

51* * * LITERATURE(CITED(

Adler,*T.*B.,*&*Rose,*G.*J.*(1998).*LongXterm*temporal*integration*in*the*anuran*auditory* system.*Nat%Neurosci,*1(6),*519–523.*

Adler,*T.*B.,*&*Rose,*G.*J.*(2000).*Integration*and*recovery*processes*contribute*to*the* temporal*selectivity*of*neurons*in*the*midbrain*of*the*northern*leopard*frog,*Rana* pipiens.*Journal%of%Comparative%Physiology%A:%Sensory,%Neural,%and%Behavioral% Physiology,*186(10),*923–937.*

Andersson,*M.*(1994).*Sexual%Selection.*Princeton,*New*Jersey:*Princeton*University* Press.*

Arak,*A.,*&*Enquist,*M.*(1993).*Hidden*preferences*and*the*evolution*of*signals.* Philosophical%Transactions:%Biological%Sciences,*340(1292),*207–213.*

Bee,*M.*A.*(2008).*Parallel*female*preferences*for*call*duration*in*a*diploid*ancestor*of* an*allotetraploid*treefrog.*Anim%Behav,*76(3),*845–853.**

Brown,*W.*D.,*Smith,*A.*T.,*Moskalik,*B.,*&*Gabriel,*J.*(2006).*Aggressive*contests*in* house*crickets:*size,*motivation*and*the*information*content*of*aggressive*songs.* Animal%Behaviour,*72(1),*225–233.*

Cotton,*S.,*Fowler,*K.,*&*Pomiankowski,*A.*(2004).*Do*sexual*ornaments*demonstrate* heightened*conditionXdependent*expression*as*predicted*by*the*handicap* hypothesis?*Proceedings%of%the%Royal%Society%of%London.%Series%B:%Biological% Sciences,*271(1541),*771–783.*

Edwards,*C.*J.,*Alder,*T.*B.,*&*Rose,*G.*J.*(2002).*Auditory*midbrain*neurons*that*count.* Nat%Neurosci,*5(10),*934–936.*

Edwards,*C.*J.,*Leary,*C.*J.,*&*Rose,*G.*J.*(2007).*Counting*on*inhibition*and*rateX dependent*excitation*in*the*auditory*system.*Journal%of%Neuroscience,*27(49),* 13384–13392.*

Endler,*J.*A.,*&*Basolo,*A.*L.*(1998).*Sensory*ecology,*receiver*biases*and*sexual* selection.*Trends%in%Ecology%and%Evolution,*13(10),*415–420.*

Gerhardt,*H.*C.*(2005a).*Acoustic*spectral*preferences*in*two*cryptic*species*of*grey* treefrogs:*implications*for*mate*choice*and*sensory*mechanisms.*Anim%Behav,* 70(1),*39–48.*

52* * * Gerhardt,*H.*C.*(2005b).*AdvertisementXcall*preferences*in*diploidXtetraploid*treefrogs* (Hyla*chrysoscelis*and*Hyla*versicolor):*implications*for*mate*choice*and*the* evolution*of*communication*systems.*Evolution,*59(2),*395–408.*

Gerhardt,*H.*C.,*&*Huber,*F.*(2002).*Acoustic%Communication%in%Insects%and%Anurans.* Chicago:*The*University*of*Chicago*Press.*

Gerhardt,*H.*C.,*Humfeld,*S.*C.,*&*Marshall,*V.*T.*(2007).*Temporal*order*and*the* evolution*of*complex*acoustic*signals.*Proceedings%of%the%Royal%Society%B:%Biological% Sciences,*274(1619),*1789–1794.*

Gerhardt,*H.*C.,*Tanner,*S.*D.,*Corrigan,*C.*M.,*&*Walton,*H.*C.*(2000).*Female*preference* functions*based*on*call*duration*in*the*gray*tree*frog*(Hyla*versicolor).*Behavioral% Ecology,*11(6),*663–669.*

Henderson,*J.*J.,*&*Gerhardt,*H.*C.*(2013).*Restoration*of*call*attractiveness*by*novel* acoustic*appendages*in*grey*treefrogs.*Animal%Behaviour,*86(3),*537–543.**

Holloway,*A.*K.,*Cannatella,*D.*C.,*Gerhardt,*H.*C.,*&*Hillis,*D.*M.*(2006).*Polyploids*with* different*origins*and*ancestors*form*a*single*sexual*polyploid*species.*The%American% Naturalist,*167(4),*E88–101.*

Johnstone,*R.*A.*(1995).*Sexual*selection,*honest*advertisement*and*the*handicap* principle:*Reviewing*the*evidence.*Biological%Reviews%of%the%Cambridge% Philosophical%Society,*70(1),*1–65.*

McComb,*K.,*Moss,*C.,*Sayialel,*S.,*&*Baker,*L.*(2000).*Unusually*extensive*networks*of* vocal*recognition*in*African*elephants.*Animal%Behaviour,*59(6),*1103–1109.**

Ptacek,*M.*B.,*Gerhardt,*H.*C.,*&*Sage,*R.*D.*(1994).*Speciation*by*polyploidy*in*treefrogs:* multiple*origins*of*the*tetraploid,*Hyla*versicolor.*Evolution,*48(3),*898–908.**

Radford,*A.*N.*(2005).*GroupXspecific*vocal*signatures*and*neighbourXstranger* discrimination*in*the*cooperatively*breeding*green*woodhoopoe.*Animal% Behaviour,*70(5),*1227–1234.*

Reichert,*M.*S.,*&*Gerhardt,*H.*C.*(2014).*Behavioral*strategies*and*signaling*in* interspecific*aggressive*interactions*in*gray*tree*frogs.*Behavioral%Ecology%,*25%(3*),* 520–530.*

Rose,*G.*J.,*Leary,*C.*J.,*&*Edwards,*C.*J.*(2011).*IntervalXcounting*neurons*in*the*anuran* auditory*midbrain:*factors*underlying*diversity*of*interval*tuning.*J%Comp%Physiol%A% Neuroethol%Sens%Neural%Behav%Physiol,*197(1),*97–108.* 53* * * Ryan,*M.*J.,*Bernal,*X.*E.,*&*Rand,*A.*S.*(2010).*Female*mate*choice*and*the*potential*for* ornament*evolution*in*túngara*frogs*Physalaemus*pustulosus.*Current%Zoology,* 56(3),*343–357.*

Ryan,*M.*J.,*&*Rand,*A.*S.*(1993).*Sexual*selection*and*signal*evolution:*the*ghost*of* biases*past.*Philosophical%Transactions%of%the%Royal%Society%B:%Biological%Sciences,* 340(1292),*187–195.*

Schul,*J.,*&*Bush,*S.*L.*(2002).*NonXparallel*coevolution*of*sender*and*receiver*in*the* acoustic*communication*system*of*treefrogs.*Proceedings%of%the%Royal%Society%B:% Biological%Sciences,*269(1502),*1847–1852.*

Schwartz,*J.*J.,*Huth,*K.,*Hunce,*R.,*&*Lentine,*B.*(2010).*Effect*of*anomalous*pulse*timing* on*call*discrimination*by*females*of*the*gray*treefrog*(Hyla*versicolor):*behavioral* correlates*of*neurobiology.*J%Exp%Biol,*213(12),*2066–2072.*

Schwartz,*J.*J.,*Huth,*K.,*Jones,*S.*H.,*Brown,*R.,*Marks,*J.,*&*Yang,*X.*(2010).*Tests*for*call* restoration*in*the*gray*treefrog*Hyla*versicolor.*Bioacoustics,*20,*59–86.*

Seeba,*F.,*Schwartz,*J.*J.,*&*Bee,*M.*A.*(2010).*Testing*an*auditory*illusion*in*frogs:* Perceptual*restoration*or*sensory*bias?*Anim%Behav,*79(6),*1317–1328.**

Wilczynski,*W.,*&*Endepols,*H.*(2006).*Central*auditory*pathways*in*anuran*amphibians:* the*anatomical*basis*of*hearing*and*sound*communication.*In*P.*M.*Narins,*A.*S.* Feng,*R.*R.*Fay,*&*A.*N.*Popper*(Eds.),*Hearing%and%Sound%Communication%in% Amphibians*(pp.*221–249).*Springer.!

54* * * !

Figure(3:1:(Examples*of*calls*used.*Shown*are*the*standard*call*(a),*gap*calls*with*one*(b)* or*two*(c)*dropped*pulses,*gap*calls*with*a*standard*appendage*(d),*a*loud*appendage* (e),*a*long*appendage*(f),*or*an*appendage*in*the*leading*position*(g).*All*appendages* shown*were*separated*from*the*pulsed*portion*of*the*call*by*50*ms.(

!

!

!

!

!

!

!

.

55* * * Figure(3:2:*Proportion*of*females*choosing*each*alternative*when*a*standard* advertisement*call*was*paired*against*a*call*with*one*dropped*pulse*or*two*dropped* pulses.*Error*bars*are*95%*credible*intervals*around*the*proportion*of*females*choosing* the*alternative.*Females*only*discriminated*against*a*call*with*two*dropped*pulses*(twoX tailed*binomial*test;***indicates*P*<*0.05;*N*=*20*for*all*tests).

56* * * Figure(3:3:*Choices*of*females*in*tests*of*a*standard*advertisement*call*versus*an* alternative.*The*noXappendage*alternative*was*the*gap*call*with*two*dropped*pulses* from*Figure*2.*Regular*(160*ms*duration,*85*dBL*SPL)*and*long*appendages*(485*ms,*85* dB*SPL)*were*ineffective*in*restoring*attractiveness.*Loud*appendages*(160*ms,*91*dB* SPL)*did*restore*attractiveness*(twoXtailed*binomial*test,***indicates*P*<*0.05).*95%* credible*intervals*shown*around*the*proportion*choosing*the*alternative.*N*=*20*for*all* tests.

57* * * Figure(3:4:**Temporal*order*of*the*appendage*relative*to*the*pulsed*portion*of*the*call* affected*attractiveness.*Females*discriminated*against*the*alternative*that*included*an* appendage*(91*dB*SPL*fast*RMS,*160*ms*duration)*in*the*leading*position*(twoXtailed* binomial*test,***indicates*P*<*0.05).*In*this*series,*the*appendage*is*separated*from*the* pulsed*portion*by*50*ms.*The*noXappendage*alternative*was*the*gap*call*with*two* dropped*pulses*from*Figure*2.*95%*credible*intervals*shown*around*the*proportion* choosing*the*alternative.*N*=20*for*all*tests.

58* * * Figure(3:5:(The*proportion*of*female*choices*in*a*direct*test*of*a*call*containing*either*a* following*or*leading*appendage;*the*silent*interval*between*the*pulsed*call*and*the* appendage*was*50*ms.*Error*bars*are*95%*credible*intervals*shown*around*the* proportion*choosing*the*leading*appendage*call.*There*was*no*significant*difference* between*the*two*alternatives*(twoXtailed*binomial*test,*P*=*0.824,*N*=*20).

59* * * !

( Figure(3:6:*When*the*silent*interval*between*the*pulsed*portion*and*the*appendage*was* reduced*to*20*ms,*the*restoration*effects*at*50*ms*were*reversed.*(a)*Following* appendages*were*ineffective*at*restoring*attractiveness,*while*(b)*leading*appendages* rescued*the*otherwise*unattractive*gap*call.*The*noXappendage*alternative*was*the*gap* call*with*two*dropped*pulses*from*Figure*2.*Error*bars*show*95%*credible*intervals* shown*around*the*proportion*choosing*the*alternative.*TwoXtailed*binomial*test,*** indicates*P*<*0.05,*N*=*20*for*all*tests.( 60* * * CHAPTER.4..

GENETIC.DIFFERENTATION.OF.POPULATIONS.OF.THE.COPE’S.GREY. TREEFROG,.HYLA%CHRYSOSCELIS%

Jennifer*J.*Henderson,*H.*Carl*Gerhardt,*Lori*S.*Eggert*

Division*of*Biological*Sciences,*University*of*Missouri,*Columbia,*MO*65211*

*

61* * * Introduction(

Multiple*patterns*of*genetic*and*phenotypic*divergence*have*been*documented* in*comparisons*of*a*single*species*with*a*large*geographic*range*(Coyne*&*Orr,*2004;*

Tregenza,*2002).*Such*divergence*at*the*genetic*level*is*not*however*always*congruent* with*phenotypic*traits*including*morphology*and*behavior.*Sometimes*genetic* differences*are*discovered*when*there*is*little*difference*in*these*phenotypic*traits,*and* in*other*systems,*phenotypic*differences*do*not*correspond*to*significant*genetic* divergence.*In*túngara*frogs,*phenotypic*isolation*(which*includes*call*characteristics* such*as*pulses*in*the*whine,*frequency*of*the*chuck,*and*duration*of*the*call,*as*well*as* ecological*niche*partitioning)*was*best*explained*by*geographic*distance*(isolation*by* distance)*rather*than*genetic*distance*(Pröhl*et%al.,*2006;*Pröhl*et%al.,*2010;*Ryan*et%al.,*

1996).*In*addition,*the*molecular*analyses*used*to*determine*genetic*divergence*may* strongly*affect*interpretations*of*behavioral*isolation*and*speciation.*For*instance,*by* comparing*genetic*divergence*using*both*mitochondrial*and*nuclear*DNA*in*the*canyon* treefrog*(Hyla%arenicolor),*Klymus*et%al.*(2010)*found*that*patterns*of*nuclear*DNA*and* call*variation*were*more*similar*than*patterns*based*on*mitochondrial*DNA.*Their*study* revealed*introgression*of*the*mitochondrial*genome*of*H.%wrightorum*into*H.%arenicolor* and*added*to*the*evidence*that*data*from*multiple*genes,*ideally*including*nuclear* genes,*are*needed*for*withinXspecies*studies*of*divergence*in*genetic*and*behavioral* patterns.*

Population*differentiation*within*H.%chrysoscelis*has*been*determined*using* multiple*methods.*One*of*the*earliest*studies*used*immunological*analysis*to*delineate*

62* * * the*species*into*eastern*and*western*groups*(Maxson*et%al.,*1977).*Subsequent*studies* using*allozymes*(Ralin*et%al.,*1983;*Ralin*&*Selander,*1979),*the*location*of*the*nucleolar* organizing*region*(Wiley*&*Little,*2000),*and*mitochondrial*DNA*(Holloway*et%al.,*2006;*

Ptacek*et%al.,*1994)*also*found*evidence*of*genetic*differentiation*between*populations* from*the*eastern*and*western*parts*of*the*species’*range.*Though*the*earliest*studies* had*limited*geographic*coverage,*studies*using*mtDNA*covered*much*of*the*species’* range*and*found*different*lineages*in*close*proximity*within*the*same*state*(*examples* in*Kansas,*Missouri,*Texas)*(Holloway*et%al.,*2006;*Ptacek*et%al.,*1994).*

Determining*the*levels*of*genetic*differentiation*in*H.%chrysoscelis*and*the* distributions*of*possible*lineages*provides*a*needed*framework*for*testing*hypotheses* about*the*origins*of*the*tetraploid*lineages*of*H.%versicolor,*which*were*derived*from* hybridization*between*lineages*of*H.%chrysoscelis%or*closely*related*species*(Holloway*et% al.,*2006).*Such*research*can*also*help*to*more*fully*understand*the*genetic*factors*that* contribute*to*behavioral*differences*between*populations*of*H.%chrysoscelis.*Males*from* the*eastern*part*of*the*range*(Georgia,*South*Carolina)*differed*from*those*in*the*west*

(Texas)*in*both*pulse*rate*and*call*duration*and*Gerhardt*(1974)*found*that*females* from*eastern*populations*discriminate*in*favor*of*local*male*call*types.*Differences*in* female*preference*between*populations*have*also*been*found*in*follow*up*studies*

(Gerhardt*et%al.,*2007;*Gerhardt,*1994).*While*the*advertisement*call*of*the*species* contains*two*frequency*peaks*(near*1.1*and*2.2*kHz),*when*presented*with*stimuli*that* only*contained*one*of*the*peaks,*female*H.%chrysoscelis*from*populations*in*Missouri*

(eastern*mtDNA*lineage)*preferred*the*lowXfrequency*alternative*and*females*from*

63* * * Minnesota*(western*mtDNA*lineage)*preferred*the*highXfrequency*alternative.*

Additionally,*when*novel*acoustic*elements*are*added*to*the*advertisement*call,* females*from*Minnesota*preferred*novel*elements*at*the*beginning*of*the*call,*while* females*from*Missouri*only*rarely*did*so*(Gerhardt*et%al.,*2007;*Seeba*et%al.,*2010).*It*is* currently*unknown*whether*these*phenotypic*differences*correlate*to*genotypic* differences.*

We*developed*and*optimized*eight*microsatellite*loci*to*examine*genetic* differentiation*throughout*the*range*of*the*species.*These*quicklyXevolving*neutral* markers*are*especially*suitable*for*this*system*because*lineages*of*H.%chrysoscelis* diverged*approximately*558,000*years*ago*(Ralin*et*al.,*1983b).*We*sampled*regions* that*may*be*contact*zones*between*the*lineages*and*identified*populations*of*H.% chrysoscelis*in*western*Missouri*that*shared*similar*morphological*features*as* populations*in*Minnesota*(J.*Henderson,*personal*observation).*Individuals*from*

Minnesota*are*typically*small*in*size*and*have*smooth,*bright*green*coloration* compared*to*the*large,*more*warty*frogs*found*in*eastern*Missouri,*where*a*large* proportion*of*individuals*are*gray.*Thus,*we*sought*to*identify*genetic*lineages*of*H.% chrysoscelis*using*nuclear*markers*to*compare*their*geographic*distributions*with*those* of*previously*identified*mtDNA*lineages.*One*expectation*was*that*some*regions*or*even* populations*contain*both*eastern*and*western*lineages.*Finally,*our*study*provides* additional*evidence*consistent*with*distinctive*genetic*lineages*in*a*wide*range*of*other* taxa.*Unique*genetic*lineages*located*in*the*Ozark*region*have*been*identified*in*many* species*including*salamanders*(Church*et%al.,*2003;*Feist*et%al.,*2014),*mussels*(Inoue*et%

64* * * al.,*2014),*and*bears*(Puckett*et%al.,*2014).*The*populations*in*this*area*may*have*been* isolated*as*a*glacial*refuge*during*the*Pleistocene,*and*in*many*species,*this*region*is*a* suture*zone*where*expanded*eastern*and*western*lineages*meet*(Rissler*&*Smith,*2010;*

Swenson*&*Howard,*2005;*Swenson,*2010).*

Methods(

Sample'Collection'and'DNA'Extraction'

Tissue*samples*were*collected*from*21*populations*during*the*2010*to*2014* breeding*seasons*(Fig.*4X1).*Samples*were*collected*as*toe*clips*or*leg*muscle*and*stored* in*95%*ethanol*and*frozen*until*DNA*extraction.*Extraction*of*DNA*was*done*using*an*

InstaGene*Matrix*(BioRad*Laboratories,*Hercules,*CA,*USA)*protocol*modified*from*

Eggert,*Maldonado,*&*Fleischer*(2005).*A*1X2*mm*tissue*sample*was*macerated*using*a* singleXedge*razor*then*placed*in*a*sterile*1.5*mL*microcentrifuge*tube*containing*250*µL* of*InstaGene*Matrix.*The*sample*was*vortexed*for*10*seconds*and*incubated*at*60°C*for*

2*hours,*vortexed*again*for*10*seconds,*and*incubated*at*100°C*for*20*minutes.*The* sample*was*then*centrifuged*at*13,000*rpm*for*3*minutes*and*the*supernatant* containing*the*DNA*transferred*to*a*sterile*tube.*All*samples*were*stored*at*X20°C*while* not*in*use.*

Genotyping'

Primers*for*eight*polymorphic*microsatellite*loci*(Table*4X1)*were*designed*and* optimized*based*on*published*sequences*(Krenz*et%al.,*1999).*Two*multiplex*polymerase* chain*reactions*(PCRs)*were*designed*for*use*with*the*Qiagen*Multiplex*PCR*kit*(Qiagen,*

Inc.,*Valencia,*CA,*USA)*(Table*4X1).*Total*volume*of*each*PCR*reaction*was*8*µL*

65* * * containing:*4*µL*Master*Mix,*0.45*µL*of*primer*mix*normalized*to*a*concentration*of*100*

µM,*0.8*µL*QXsolution,*and*0.5*µL*DNA.*The*forward*primer*for*each*locus*was*endX labeled*with*florescent*dye.*Both*multiplexes*had*PCR*conditions*of*15*minutes*at*95°C*

(initial*denaturation)*followed*by*40*cycles*of*30*seconds*denaturation*at*94°C,*primer* annealing*at*57.1°C*(multiplex*1)*or*59°C*(multiplex*2)*for*90*seconds,*and*primer* extension*at*72°C*for*60*seconds,*and*a*final*extension*step*of*60°C*for*30*minutes.*A* negative*(no*DNA)*control*sample*was*included*with*all*reactions*to*test*for*reagent* contamination.*Amplification*products*were*visualized*on*a*2%*agarose*gel*before* submission*for*fragment*analysis*in*an*ABI*3730*DNA*Analyzer*at*the*University*of*

Missouri*DNA*Core*Facility*(Columbia,*MO)*using*Liz*600*lane*standard.*Allele*scoring*for* each*individual*was*performed*using*GENEMARKER*software*(v1.97,*Softgenetics,*LLC,*

State*College,*PA).*

Population'Genetic'Analyses'

We*tested*for*deviations*from*expected*heterozygosity*values*under*HardyX

Weinberg*equilibrium*(HWE)*and*for*linkage*disequilibrium*for*each*population*in*

GENEPOP*v4.2*(Raymond*&*Rousset,*1995;*Rousset,*2008).*We*applied*a*Bonferroni* correction*(α*<*0.006)*for*all*comparisons.*In*GENEPOP,*we*calculated*the*expected*(HE)* and*observed*(HO)*heterozygosity*values*and*the*mean*number*of*alleles*(A).*To*correct* for*unequal*sample*sizes,*we*calculated*rarefied*allelic*richness*(Ar)*and*private*allelic* richness*(Arp)*in*HPXRARE*(Kalinowski,*2005).*Population*differentiation*(FST)*and* significance*levels*were*determined*in*ARLEQUIN*v3.5*(Excoffier*&*Lischer,*2010).*The*

66* * * FST*matrix*was*used*in*a*Mantel*test,*along*with*the*geographic*distance*between* populations*(km),*to*test*for*isolation*by*distance*in*GENALEX*(Peakall*&*Smouse,*2006).*

We*analyzed*population*structure*in*STRUCTURE*v2.3*(Pritchard*et%al.,*2000),* applying*the*no*admixture*model*assuming*correlated*frequencies.*We*performed*10* repetitions*of*each*independent*cluster*(K)*value*between*one*and*27,*with*a*burnin*of*

250,000*iterations*followed*by*750,000*MCMC*iterations.*Models*for*the*replicates* were*averaged*in*CLUMPP*v1.2*(Jakobsson*&*Rosenberg,*2007),*and*models*for*two*and* three*clusters*were*displayed*using*DISTRUCT*v1.1*(Rosenberg,*2004).*We*identified*the* number*of*clusters*with*the*highest*value*of*ΔK*(Evanno*et%al.,*2005)*with*the*aid*of*

STRUCTURE*HARVESTER*(Earl*&*VonHoldt,*2012)*and*determined*this*to*be*the*number* of*genetic*clusters*given*the*data.*

Results(

We*genotyped*97*samples*of*H.%chrysoscelis%from*21*populations*(Table*4X2).*

Two*of*the*8*microsatellite*loci*deviated*from*HWE*at*the*Bonferroni*corrected*α*<*

0.006.*Locus*6M1K*deviated*in*the*VA*population;*locus*6M2B*deviated*in*the*Jackson*

County,*MO*(J),*Phelps*County,*MO*(P),*and*Oregon*County,*MO*(O)*populations.*Since* we*did*not*observe*evidence*that*any*locus*deviated*from*expectations*under*HWE* broadly*across*populations,*we*retained*all*loci*in*our*analyses.*There*was*no*evidence* for*linkage*between*pairs*of*loci*in*any*population.*Observed*heterozygosity*in*each* population*ranged*from*0.250*to*0.762*(mean*=*0.602,*standard*deviation*=*0.130).*The* mean*number*of*alleles,*corrected*for*sample*size*using*rarefaction*(Arp),*and*private* allelic*richness*were*similar*across*all*populations*(Table*4X2).*

67* * * Population'Structure'

Pairwise*values*of*FST*ranged*from*X0.027*(a*value*not*significantly*different*from*

0)*to*0.667*(Table*4X3).*Analysis*in*STRUCTURE*identified*three*genetic*clusters:*Eastern,*

Ozark,*and*Western*lineages*(Fig.*4X2).*There*was*a*substantial*increase*in*delta*K*for*K*=*

3*(delta*K*=*140.263)*over*K*=2*(delta*K*=*24.759)*(Supplemental*Table*4X1).*Although* three*genetic*clusters*(K)*were*best*supported*by*our*data,*at*K*=*2*we*recaptured*the*

Eastern*and*Western*lineages*as*defined*using*mtDNA*(Ptacek*et%al.,*1994)*(Fig.*4X2,*4X

3).*We*reran*FST*tests*after*grouping*samples*into*the*three*clusters*identified*in*

STRUCTURE.*All*comparisons*showed*significant*differentiation*at*the*α*<*0.017*level*

(Table*4X4).*

Isolation'by'Distance(

We*analyzed*the*relationship*between*geographic*distance*(km)*and*genetic* distance*(FST)*between*the*9*populations*sampled*in*Missouri.*We*only*performed*the* analysis*on*this*subset*of*samples*as*we*had*a*fairly*continuous*sample*across*the*state* and*previous*studies*using*mtDNA*results*(Ptacek*et%al.,*1994)*suggest*both*genetic* lineages*come*in*contact*in*this*area.*No*significant*correlation*between*genetic*and* geographic*distance*was*observed*using*the*Mantel*test*performed*in*GENALEX*(P*=*

0.481).*

Discussion*

Previous*molecular*methods*identified*two*distinct*genetic*lineages*of*H.% chrysoscelis,*one*located*in*the*eastern*part*of*the*species’*range*(Kentucky,*Alabama,*

Georgia,*South*Carolina,*etc.)*and*one*in*the*western*region*(Minnesota,*Oklahoma,*

68* * * Texas)*(Holloway%et%al.,*2006;*Ptacek*et%al.,*1994;*Ralin*et%al.,*1983;*Ralin*&*Selander,*

1979;*Romano*et%al.,*1987).*Using*microsatellite*loci,*we*identified*a*new*Ozark*lineage* that*is*distinct*from*the*previously*reported*eastern*and*western*lineages.*Our*sampling* of*Missouri*locales*was*the*most*extensive*and*for*some*parts*of*the*range,*our*sample* sizes*were*very*low.*However,*despite*previous*studies*and*the*extensive*work*on*this* species,*identification*of*fineXscale*differentiation*has*not*been*identified*in*any*other* part*of*the*range.*The*identification*of*a*third*lineage*of*H.%chrysoscelis*may*be* important*in*understanding*the*relationship*between*the*diploid*H.%chrysoscelis*and*the* tetraploid*H.%versicolor.*While*speciation*due*to*polyploidy*is*relatively*rare*in*animal* species,*H.%versicolor*is*considered*to*be*a*single*group*of*interbreeding*tetraploids*that* arose*from*three*independent*alloploidy*events*from*three*diploid*ancestors*(Holloway* et%al.,*2006).*These*diploid*ancestors*are*thought*to*be*the*extant*H.%chrysoscelis*and* two*extinct*diploid*species.*The*previously*unidentified*Ozark*lineage*may*provide* important*information*about*the*diploid*ancestors*and*unique*origins*of*the*tetraploid*

H.%versicolor.*

We*also*identified*regions*in*Missouri*and*Texas*in*which*different*lineages*of*H.% chrysoscelis*are*in*close*geographic*contact.*While*the*species’*range*covers*most*of*the* eastern*continental*United*States,*there*are*regions*of*Missouri*and*Texas*that*contain* only*the*tetraploid*H.%versicolor%(Romano*et%al.,*1987;*H.C*Gerhardt,*personal* observation).*These*“wedges”*of*allopatric*H.%versicolor%may*act*as*a*barrier*between* lineages*of*H.%chyroscelis,*and*thus*in*some*cases,*we*can*find*distinct*genetic*lineages* less*than*100*km*apart.*Further*studies*are*needed*to*delineate*what*factors,*such*as*

69* * * landscape*features*or*habitat*occupation,*may*contribute*to*isolation*between* populations*of*H.%chrysoscelis*and*H.%versicolor.*

The*genetic*lineages*identified*in*this*study*were*congruent*with*subtle*but*wellX documented*behavioral*differences.*Populations*in*Minnesota*exhibit*different* preferences*for*call*frequency*and*novel*complex*calls*compared*to*populations*from* eastern*Missouri*(Gerhardt*et%al.,*2007;*Gerhardt*et%al.,*2007;*Seeba*et%al.,*2010;*

Schrode*et%al.,*2012;*M.A.*Bee,*unpublished*data).*Our*results*also*identified* populations*in*western*Missouri*that*are*of*the*same*genetic*lineage*as*Minnesota*H.% chrysoscelis.*It*will*be*important*for*future*experiments*to*examine*the*behavioral* preferences*of*these*western*Missouri*populations*to*determine*whether*behavioral* divergence*correlates*with*genetic*and/or*geographic*distance.*In*wideXranging*species,* phenotypic*divergence*has*been*correlated*with*geographic*distance*(Pröhl*et%al.,*2006),* genetic*distance*(Wang*&*Summers,*2010),*or*both*(Irwin*et%al.,*2008).*

Our*study*also*adds*to*the*evidence*that*the*Ozark*region*(southern*Missouri* and*northern*Arkansas*area)*contains*distinctive*genetic*lineages*in*a*wide*range*of* taxa.*For*many*species,*including*salamanders*(Church*et%al.,*2003),*bobcats*(Reding*et% al.,*2012),*and*frogs*(this*study),*the*Ozark*region*is*a*contact*zone*between*eastern*and* western*lineages.*Hall*&*Kelson*(1959)*attributed*to*this*common*pattern*of*distinct* eastern*and*western*sister*taxa*to*the*aridification*of*the*region*during*the*Pleistocence* interglacial*periods,*which*separated*many*species*into*eastern*and*western* populations.*These*populations*acted*as*refugia,*isolated*from*each*other*by*unsuitable* habitat*between*them.*This*explanation*has*been*supported*with*patterns*of*genetic*

70* * * structure,*where*ancestral*genotypes*are*restricted*to*the*edges*of*the*USA*and*absent* from*the*central*region*(Reding*et%al.,*2012).*This*is*consistent*with*the*expectation*that* ancestral*genotypes*center*on*the*origins*of*range*expansion*(Templeton,*2006).*When* ancestral*ranges*expand,*the*Great*Plains*and*Ozark*regions*are*often*where*lineages* come*into*contact,*creating*a*suture*zone*for*many*North*American*species*(Swenson*&*

Howard,*2005;*Swenson,*2010).*

In*addition*to*being*a*suture*zone*for*many*taxa,*the*Ozark*region*often*has* unique*populations*endemic*to*the*area.*Highly*differentiated*populations*in*this*area* have*been*reported*in*multiple*taxa*(Church*et%al.,*2003;*Feist%et%al.,*2014;*Inoue*et%al.,*

2014;*Mayden,*1985;*Puckett*et%al.,*2014;*this*study).*For*many*species,*eastern*and* western*lineages*have*diverged*and*reXestablished*contact*in*the*Ozark*region.*

Populations*in*this*central*area*may*have*been*isolated*from*other*lineages*during*the*

Pleistocene*glaciations,*leading*to*divergence*(Mayden,*1988).*In*the*eastern*tiger* salamander,*for*example,*eastern*and*western*lineages*diverged*up*to*two*million*years* ago,*and*during*Pleistocene*glaciations,*disjunct*populations*in*small*refugia*were* isolated*from*other*populations*for*200,000X500,000*years*(Church*et%al.,*2003).*Unique* populations*in*the*central*USA*region*may*also*represent*remnant*lineages*that*have* remained*isolated*from*contemporary*populations,*such*as*in*the*case*of*black*bears*in* parts*of*Missouri*(Puckett*et%al.,*2014).*Thus,*this*pattern*of*unique*populations*located* in*this*region*may*have*important*conservation*implications*for*many*species.*

For*H.%chrysoscelis,*future*studies*should*examine*the*extent*to*which*behavioral* differentiation*corresponds*to*the*observed*genetic*differentiation.*The*differences*in*

71* * * female*preference*previously*mentioned*involved*geographically*distant*populations*

(Gerhardt*et%al.,*2007;*Gerhardt*et%al.,*2007;*Seeba*et%al.,*2010*).*Does*this* differentiation*correlate*with*differences*between*western*and*eastern*or*western*and*

Ozark*genetic*lineages?***In*Missouri,*we*identified*populations*that*are*less*than*100* km*apart*that*belong*to*different*lineages.*Comparisons*of*preference*for*populations* that*are*geographically*and/or*genetically*distant*across*the*species*range*can*tease* apart*the*effect*of*isolation*by*distance*on*behavioral*phenotypes.

72* * * LITERATURE(CITED*

Church,*S.*a,*Kraus,*J.*M.,*Mitchell,*J.*C.,*Church,*D.*R.,*&*Taylor,*D.*R.*(2003).*Evidence* for*multiple*Pleistocene*refugia*in*the*postglacial*expansion*of*the*eastern*tiger* salamander,*Ambystoma*tigrinum*tigrinum.*Evolution;%International%Journal%of% Organic%Evolution,*57(2),*372–83.*

Coyne,*J.*A.,*&*Orr,*H.*A.*(2004).*Speciation.*Sunderland,*MA:*Sinauer*Associates.*

Earl,*D.*A.,*&*VonHoldt,*B.*M.*(2012).*STRUCTURE*HARVESTER:*a*website*and*program* for*visualizing*STRUCTURE*output*and*implementing*the*Evanno*method.* Conservation%Genetics%Resources,*4(2),*359–361.*

Eggert,*L.*S.,*Maldonado,*J.*E.,*&*Fleischer,*R.*C.*(2005).*Nucleic*Acid*Isolation*from* Ecological*Samples—Animal*Scat*and*Other*Associated*Materials.*In*and*E.*H.*R.*B.* T.XM.*in*E.*Elizabeth*A.*Zimmer*(Ed.),*Molecular%Evolution:%Producing%the% Biochemical%Data*(Vol.*Volume*395,*pp.*73–82).*Academic*Press.**

Evanno,*G.,*Regnaut,*S.,*&*Goudet,*J.*(2005).*Detecting*the*number*of*clusters*of* individuals*using*the*software*STRUCTURE:*a*simulation*study.*Molecular%Ecology,* 14(8),*2611–2620.*

Excoffier,*L.,*&*Lischer,*H.*(2010).*Arlequin*suite*ver*3.5:*a*new*series*of*programs*to* perform*population*genetics*analyses*under*Linux*and*Windows.*Molecular%Ecology% Resources,*10(3),*564–567.*

Feist,*S.*M.,*Briggler,*J.*T.,*Koppelman,*J.*B.,*&*Eggert,*L.*S.*(2014).*WithinXriver*gene*flow* in*the*hellbender*(Cryptobranchus*alleganiensis)*and*implications*for*restorative* release.*Conservation%Genetics,*15(4),*953–966.*

Gerhardt,*H.*C.*(1974).*Mating*call*differences*between*eastern*and*western* populations*of*the*treefrog*Hyla*chrysoscelis.*Copeia,*1974(2),*534–536.*

Gerhardt,*H.*C.*(1994).*Reproductive*character*displacement*of*female*mate*choice*in* the*grey*treefrog,*Нуla*chrysoscelis.*Anim%Behav,*47,*959–969.*

Gerhardt,*H.*C.,*Humfeld,*S.*C.,*&*Marshall,*V.*T.*(2007).*Temporal*order*and*the* evolution*of*complex*acoustic*signals.*Proceedings%of%the%Royal%Society%B:%Biological% Sciences,*274(1619),*1789–1794.*

73* * * Gerhardt,*H.*C.,*MartínezXRivera,*C.*C.,*Schwartz,*J.*J.,*Marshall,*V.*T.,*&*Murphy,*C.*G.* (2007).*Preferences*based*on*spectral*differences*in*acoustic*signals*in*four*species* of*treefrogs*(Anura:*Hylidae).*J%Exp%Biol,*210(17),*2990–2998.**

Hall,*E.,*&*Kelson,*K.*(1959).*The%Mammals%of%North%America.*New*York:*Ronald*Press.*

Holloway,*A.*K.,*Cannatella,*D.*C.,*Gerhardt,*H.*C.,*&*Hillis,*D.*M.*(2006).*Polyploids*with* different*origins*and*ancestors*form*a*single*sexual*polyploid*species.*The%American% Naturalist,*167(4),*E88–101.**

Inoue,*K.,*Monroe,*E.*M.,*Elderkin,*C.*L.,*&*Berg,*D.*J.*(2014).*Phylogeographic*and* population*genetic*analyses*reveal*Pleistocene*isolation*followed*by*high*gene*flow* in*a*wide*ranging,*but*endangered,*freshwater*mussel.*Heredity,*112(3),*282–90.**

Irwin,*D.*E.,*Thimgan,*M.*P.,*&*Irwin,*J.*H.*(2008).*Call*divergence*is*correlated*with* geographic*and*genetic*distance*in*greenish*warblers*(Phylloscopus*trochiloides):*a* strong*role*for*stochasticity*in*signal*evolution?*J%Evol%Biol,*21(2),*435–448.**

Jakobsson,*M.,*&*Rosenberg,*N.*(2007).*CLUMPP:*a*cluster*matching*and*permutation* program*for*dealing*with*label*switching*and*multimodality*in*analysis*of* population*structure.*Bioinformatics,*23(14),*1801–1806.*

Kalinowski,*S.*T.*(2005).*hpXrare*1.0:*a*computer*program*for*performing*rarefaction*on* measures*of*allelic*richness.*Molecular%Ecology%Notes,*5(1),*187–189.**

Klymus,*K.*E.,*Humfeld,*S.*C.,*Marshall,*V.*T.,*Cannatella,*D.,*&*Gerhardt,*H.*C.*(2010).* Molecular*patterns*of*differentiation*in*canyon*treefrogs*(Hyla*arenicolor):* evidence*for*introgressive*hybridization*with*the*Arizona*treefrog*(H.*wrightorum)* and*correlations*with*advertisement*call*differences.*Journal%of%Evolutionary% Biology,*23(7),*1425–1435.*

Krenz,*J.*D.,*Semlitsch,*R.*D.,*Gerhardt,*H.*C.,*&*Mahoney,*P.*A.*(1999).*Isolation*and* characterization*of*simple*sequence*repeat*loci*in*the*gray*tree*frog,*Hyla* chrysoscelis.*Genome,*42(4),*676–680.*

Maxson,*L.,*Pepper,*E.,*&*Maxson,*R.*D.*(1977).*Immunological*resolution*of*a*diploidX tetraploid*species*complex*of*tree*frogs.*Science,*197(4307),*1012–1013.*

Mayden,*R.*(1985).*Biogeography*of*Ouachita*highland*fishes.*Southwest%Naturalist,*30,* 195–211.*

Mayden,*R.*(1988).*Vicariance*biogeography,*parsimony,*and*evolution*in*North* American*freshwater*fishes.*Systematic%Biology,*37,*329–355.* 74* * * Peakall,*R.,*&*Smouse,*P.*(2006).*Genalex*6:*genetic*analysis*in*Excel.*Population*genetic* software*for*teaching*and*research.*Molecular%Ecology%Notes,*6(1),*288–295.**

Pritchard,*J.*K.,*Stephens,*M.,*&*Donnelly,*P.*(2000).*Inference*of*population*structure* using*multilocus*genotype*data.*Genetics,*155(2),*945–959.**

Pröhl,*H.,*Koshy,*R.*A.,*Mueller,*U.,*Rand,*A.*S.,*&*Ryan,*M.*J.*(2006).*Geographic* Variation*of*Genetic*and*Behavioral*Traits*in*Northern*and*Southern*Túngara*Frogs.* Evolution,*60(8),*1669–1679.**

Pröhl,*H.,*Ron,*S.*R.,*&*Ryan,*M.*J.*(2010).*Ecological*and*genetic*divergence*between* two*lineages*of*middle*American*tungara*frogs*Physalaemus*(=*Engystomops)* pustulosus.*BMC%Evol%Biol,*10,*146.*

Ptacek,*M.*B.,*Gerhardt,*H.*C.,*&*Sage,*R.*D.*(1994).*Speciation*by*polyploidy*in*treefrogs:* multiple*origins*of*the*tetraploid,*Hyla*versicolor.*Evolution,*48(3),*898–908.**

Puckett,*E.*E.,*Kristensen,*T.*V,*Wilton,*C.*M.,*Lyda,*S.*B.,*Noyce,*K.*V,*Holahan,*P.*M.,*…* Eggert,*L.*S.*(2014).*Influence*of*drift*and*admixture*on*population*structure*of* American*black*bears*(Ursus*americanus)*in*the*Central*Interior*Highlands,*USA,*50* years*after*translocation.*Molecular%Ecology,*23(10),*2414–2427.**

Ralin,*D.*B.,*Romano,*M.*A.,*&*Kilpatrick,*C.*W.*(1983a).*The*tetraploid*treefrog*Hyla* versicolor:*evidence*for*a*single*origin*from*the*diploid*H.*chrysoscelis.* Herpetologica,*39(3),*212–225.*

Ralin,*D.*B.,*Romano,*M.*A.,*&*Kilpatrick,*C.*W.*(1983b).*The*tetraploid*treefrog*Hyla* versicolor:*evidence*for*a*single*origin*from*the*diploid*H.*chrysoscelis.* Herpetologica,*39(3),*212–225.*

Raymond,*M.,*&*Rousset,*F.*(1995).*GENEPOP*(Version*1.2):*Population*Genetics* Software*for*Exact*Tests*and*Ecumenicism.*Journal%of%Heredity%,*86%(3*),*248–249.**

Reding,*D.*M.,*Bronikowski,*A.*M.,*Johnson,*W.*E.,*&*Clark,*W.*R.*(2012).*Pleistocene*and* ecological*effects*on*continentalXscale*genetic*differentiation*in*the*bobcat*(Lynx* rufus).*Molecular%Ecology,*21(12),*3078–93.**

Rissler,*L.*J.,*&*Smith,*W.*H.*(2010).*Mapping*amphibian*contact*zones*and* phylogeographical*break*hotspots*across*the*United*States.*Molecular%Ecology,* 19(24),*5404–5416.**

75* * * Romano,*M.*A.,*Ralin,*D.*B.,*Guttman,*S.*I.,*&*Skillings,*J.*H.*(1987).*Parallel*electromorph* variation*in*the*diploidXtetraploid*gray*treefrog*complex.*The%American%Naturalist,* 130(6),*864–878.*

Rosenberg,*N.*(2004).*distruct:*a*program*for*the*graphical*display*of*population* structure.*Ecology%Notes,*4(1),*137–138.*

Rousset,*F.*(2008).*genepop’007:*a*complete*reXimplementation*of*the*genepop* software*for*Windows*and*Linux.*Molecular%Ecology%Resources,*8(1),*103–106.**

Ryan,*M.*J.,*Rand,*A.*S.,*&*Weigt,*L.*A.*(1996).*Allozyme*and*Advertisement*Call*Variation* in*the*Tungara*Frog,*Physalaemus*pustulosus.*Evolution,*50(6),*2435–2453.*

Schrode,*K.*M.,*Ward,*J.*L.,*Vélez,*A.,*&*Bee,*M.*A.*(2012).*Female*preferences*for* spectral*call*properties*in*the*western*genetic*lineage*of*Cope’s*gray*treefrog*(Hyla* chrysoscelis).*Behavioral%Ecology%and%Sociobiology,*66(12),*1595–1606.**

Seeba,*F.,*Schwartz,*J.*J.,*&*Bee,*M.*A.*(2010).*Testing*an*auditory*illusion*in*frogs:* Perceptual*restoration*or*sensory*bias?*Anim%Behav,*79(6),*1317–1328.**

Swenson,*N.*G.*(2010).*Mapping*the*suturing*of*a*continental*biota.*Molecular%Ecology,* 19(24),*5324–7.**

Swenson,*N.*G.,*&*Howard,*D.*J.*(2005).*Clustering*of*contact*zones,*hybrid*zones,*and* phylogeographic*breaks*in*North*America.*The%American%Naturalist,*166(5),*581– 91.**

Templeton,*A.*R.*(2006).*Population%Genetics%and%Microevolutionary%Theory.*Hoboken,* New*Jersey:*John*Wiley*&*Sons*Inc.*

Tregenza,*T.*(2002).*Divergence*and*Reproductive*Isolation*in*the*Early*Stages*of* Speciation.*Genetica,*116,*291–300.*

Wang,*I.*J.,*&*Summers,*K.*(2010).*Genetic*structure*is*correlated*with*phenotypic* divergence*rather*than*geographic*isolation*in*the*highly*polymorphic*strawberry* poisonXdart*frog.*Molecular%Ecology,*19(3),*447–458.**

Wiley,*J.*E.,*&*Little,*M.*L.*(2000).*Replication*banding*patterns*of*the*diploidXtetraploid* treefrogs*Hyla*chrysoscelis*and*H.*versicolor.*Cytogenetics%and%Cell%Genetics,*88(1X 2),*11–14.*

!

76* * *

Table&4(4:!Pairwise!FST!for!population!pairs!using!clusters!identified!by!STRUCTURE.! Significant!values!following!Bonferroni!correction!(P!

Eastern Ozark Western Eastern - Ozark 0.055 - Western 0.074 0.064 - !

! 80! Supplementary,Table,405:!Results!of!Evanno!test!best!supporting!three!genetic!clusters.! ! K Reps Mean LnP(K) Delta K 1 10 -3984.31 2 10 -3795.05 24.759 3 10 -3634.48 140.263 4 10 -3580.28 10.765 5 10 -3501.57 10.736 6 10 -3443.96 64.719 7 10 -3513.40 1.157 8 10 -3507.36 0.077 ! !

! 81! ! ! Figure'4)1:!Locations!of!samples!collected.! !

! 82!

! Figure!4)3:#(a)#Population#lineages#inferred#using#mtDNA#cytochrome#b#sequences# (adapted#from#Holloway#et#al.,#2006).#Circles#=#Western#lineage;#Squares#=#Eastern# lineage.#(b)#Geographic#distribution#of#genetic#clusters#identified#in#STRUCTURE.# #

! 84# CHAPTER(5(

CONCLUSIONS(

Jennifer!J.!Henderson!

Division!of!Biological!Sciences,!University!of!Missouri,!Columbia,!MO!65211

85! ! ! My!research!employed!multiple!methods!to!examine!mate!choice!behavior!in! the!gray!treefrog!species!complex.!In!chapter!2,!I!examined!how!the!reported!response! properties!of!temporally!selective!neurons!might!influence!complex!call!attractiveness! in!Hyla#versicolor.#As!expected!based!on!the!response!properties!of!the!neurons,!the! removal!of!a!pulse!to!create!a!gap!of!inappropriate!length!made!a!call!unattractive!(see! also:!(Schwartz,!Huth,!Hunce,!et!al.,!2010).!Similar!to!the!findings!of!Gerhardt!et#al.!

(2007),!I!found!that!novel!acoustic!appendages!that!followed!a!train!of!pulses!were! attractive!to!females,!while!the!same!appendage!in!the!leading!position!was!not.!An! appendage!in!the!following!position!that!was!of!relatively!loud!amplitude!was!effective! in!restoring!the!attractiveness!of!a!call!that!contained!gaps.!While!these!results!are! consistent!with!the!response!properties!of!temporally!selective!neurons,!one!limitation! is!that!I!did!not!record!from!the!neurons!directly.!These!data!provide!directions!for! other!researchers!such!as!Gary!Rose,!at!the!University!of!Utah,!to!use!some!of!the!same! acoustic!stimuli!in!studies!of!single!auditory!neurons!in!the!midbrain!area!thought!to! control!acoustic!selectivity!in!these!treefrogs.!!

In!chapter!3,!I!preformed!a!similar!experiment!using!the!closely!related!species,!

H.#chrysoscelis.!While!both!species!have!a!pulsed!advertisement!call,!H.#versicolor!uses! pulse!duration,!shape,!and!interval!as!recognition!mechanisms,!while!H.#chrysoscelis! relies!on!pulse!rate!(Johannes!Schul!&!Bush,!2002).!This,!combined!with!emerging! results!from!neurophysiology!as!well!as!observed!behavioral!differences!in!response!to! complex!calls!(Gerhardt,!Humfeld,!et!al.,!2007;!Seeba!et!al.,!2010),!suggested!that!H.# chrysoscelis!may!behave!differently!than!H.#versicolor.#.#I!found!that!a!variety!of!acoustic!

86! ! ! appendages!we!able!to!restore!call!attractiveness!in!this!species.!Both!following!and! leading!appendages!could!rescue!an!otherwise!unattractive!gap!call,!and!my! experiments!highlighted!an!important!condition:!the!duration!of!the!silent!interval! between!the!pulse!train!(advertisement!call)!and!the!appendage.!Shortening!this! interval!between!the!two!call!elements!could!promote!(leading!appendage)!or!eliminate!

(following!appendages)!the!rescuing!effect.!Lengthening!the!interval!eliminated!the! restoration!by!compound!calls!with!leading!appendages!and!increased!restoration!by! compound!calls!with!following!appendages.!As!in!the!experiments!described!in!chapter!

2,!one!limitation!of!this!study!was!that!I!did!not!record!from!the!neurons.!These! recordings!would!provide!important!and!necessary!insights!to!the!mechanisms! underlying!the!behavioral!preferences.!Preliminary!results!of!systematically!altering!the! spacing!between!the!call!elements!suggest!that!more!experimental!data!of!this!kind!will! provide!a!more!information!about!what!factors!influence!complex!call!attractiveness!in! this!species!and!in!H.#versicolor.!

The!behavioral!differences!in!female!response!to!complex!calls!between! populations!of!H.#chrysoscelis!provided!some!of!the!rationale!for!chapter!4.!The! populations!used!in!behavioral!experiments!came!from!two!different!genetic!lineages!as! determined!by!mitochondrial!DNA!(Holloway!et!al.,!2006;!Ptacek!et!al.,!1994).!The!goal! of!chapter!4!was!to!determine!the!fine`scale!genetic!structure!of!H.#chrysoscelis!using! microsatellite!markers.!While!this!species!had!been!studied!extensively!using!a!variety! of!methods!(Holloway!et!al.,!2006;!Maxson!et!al.,!1977;!Ptacek!et!al.,!1994;!Ralin!et!al.,!

1983a;!Ralin!&!Selander,!1979;!Wiley!&!Little,!2000),!the!rapid!mutation!rate!of!

87! ! ! microsatellite!loci!is!especially!suitable!for!studying!genetic!structure!on!relatively!short! time`scales.!This!study!supported!the!existence!of!multiple!genetic!lineages!in!H.# chrysoscelis.!Along!with!the!previously!identified!eastern!and!western!lineages,!I! identified!a!third!lineage!located!in!the!Ozark!region!of!southern!Missouri.!In!multiple! taxa,!unique!genetic!lineages!in!this!region!have!been!reported!(Church!et!al.,!2003;!

Feist!et!al.,!2014;!Inoue!et!al.,!2014;!Puckett!et!al.,!2014).!My!data!provide!additional! support!for!the!unique!genetic!structure!found!in!this!geographic!area!as!well!as!the! location!of!a!suture!zone!between!eastern!and!western!populations!(Rissler!&!Smith,!

2010;!Swenson,!2010).!!

However,!one!limitation!of!this!study!is!the!small!sample!size!for!a!number!of!my! sampling!locations.!Populations!in!Missouri!often!had!the!highest!sample!sizes!of!my! study!and!increasing!sample!size!in!other!populations!would!strengthen!my!results.!In! addition!to!increasing!number!of!samples!per!location,!more!thorough!geographic! coverage!of!the!entire!range!of!H.#chrysoscelis!is!an!important!future!direction.!By! sampling!across!a!cline,!the!genetic!structure!could!be!compared!to!other!observed! behavioral!differences,!such!as!the!clinal!variation!in!male!calls!(Gerhardt,!1974).!Finally,! it!would!be!interesting!to!compare!the!population!structure!as!determined!by! microsatellite!loci!to!phylogenies!obtained!from!next`generation!sequencing!methods.!

Differences!in!methodology!may!provide!more!information!to!provide!a!better! comparison!between!genetic!and!behavioral!patterns.!

!

88! ! ! My!research!provides!insights!about!mate!choice!behavior!of!treefrogs!using! very!different!research!methodologies.!My!behavioral!experiments!set!the!stage!for! examining!correlations!between!behavioral!selectivity!and!auditory!mechanisms!that! are!studied!at!the!single!neuron!level!in!other!laboratories.!Additionally,!I!used!genetic! methods!to!understand!the!population!structure!and!evolutionary!history!to!contribute! to!a!better!understanding!of!observed!behavioral!differentiation.!My!work!exemplifies! how!the!integration!of!multiple!methods!can!result!in!a!more!complete!understanding! of!an!animal’s!behavior.

89! ! ! LITERATURE!CITED!

Church,!S.!a,!Kraus,!J.!M.,!Mitchell,!J.!C.,!Church,!D.!R.,!&!Taylor,!D.!R.!(2003).!Evidence! for!multiple!Pleistocene!refugia!in!the!postglacial!expansion!of!the!eastern!tiger! salamander,!Ambystoma!tigrinum!tigrinum.!Evolution;#International#Journal#of# Organic#Evolution,!57(2),!372–83.!!

Feist,!S.!M.,!Briggler,!J.!T.,!Koppelman,!J.!B.,!&!Eggert,!L.!S.!(2014).!Within`river!gene!flow! in!the!hellbender!(Cryptobranchus!alleganiensis)!and!implications!for!restorative! release.!Conservation#Genetics,!15(4),!953–966.!!

Gerhardt,!H.!C.!(1974).!Mating!call!differences!between!eastern!and!western! populations!of!the!treefrog!Hyla!chrysoscelis.!Copeia,!1974(2),!534–536.!

Gerhardt,!H.!C.,!Humfeld,!S.!C.,!&!Marshall,!V.!T.!(2007).!Temporal!order!and!the! evolution!of!complex!acoustic!signals.!Proceedings#of#the#Royal#Society#B:#Biological# Sciences,!274(1619),!1789–1794.!!

Holloway,!A.!K.,!Cannatella,!D.!C.,!Gerhardt,!H.!C.,!&!Hillis,!D.!M.!(2006).!Polyploids!with! different!origins!and!ancestors!form!a!single!sexual!polyploid!species.!The#American# Naturalist,!167(4),!E88–101.!!

Inoue,!K.,!Monroe,!E.!M.,!Elderkin,!C.!L.,!&!Berg,!D.!J.!(2014).!Phylogeographic!and! population!genetic!analyses!reveal!Pleistocene!isolation!followed!by!high!gene!flow! in!a!wide!ranging,!but!endangered,!freshwater!mussel.!Heredity,!112(3),!282–90.!!

Maxson,!L.,!Pepper,!E.,!&!Maxson,!R.!D.!(1977).!Immunological!resolution!of!a!diploid` tetraploid!species!complex!of!tree!frogs.!Science,!197(4307),!1012–1013.!

Ptacek,!M.!B.,!Gerhardt,!H.!C.,!&!Sage,!R.!D.!(1994).!Speciation!by!polyploidy!in!treefrogs:! multiple!origins!of!the!tetraploid,!Hyla!versicolor.!Evolution,!48(3),!898–908.!!

Puckett,!E.!E.,!Kristensen,!T.!V,!Wilton,!C.!M.,!Lyda,!S.!B.,!Noyce,!K.!V,!Holahan,!P.!M.,!…! Eggert,!L.!S.!(2014).!Influence!of!drift!and!admixture!on!population!structure!of! American!black!bears!(Ursus!americanus)!in!the!Central!Interior!Highlands,!USA,!50! years!after!translocation.!Molecular#Ecology,!23(10),!2414–2427.!!

Ralin,!D.!B.,!Romano,!M.!A.,!&!Kilpatrick,!C.!W.!(1983).!The!tetraploid!treefrog!Hyla! versicolor:!evidence!for!a!single!origin!from!the!diploid!H.!chrysoscelis.! Herpetologica,!39(3),!212–225.!

90! ! ! Ralin,!D.!B.,!&!Selander,!R.!K.!(1979).!Evolutionary!genetics!of!diploid`tetraploid!species! of!treefrogs!of!the!genus!Hyla.!Evolution,!33(2),!595–608.!

Rissler,!L.!J.,!&!Smith,!W.!H.!(2010).!Mapping!amphibian!contact!zones!and! phylogeographical!break!hotspots!across!the!United!States.!Molecular#Ecology,! 19(24),!5404–5416.!!

Schul,!J.,!&!Bush,!S.!L.!(2002).!Non`parallel!coevolution!of!sender!and!receiver!in!the! acoustic!communication!system!of!treefrogs.!Proceedings#of#the#Royal#Society#B:# Biological#Sciences,!269(1502),!1847–1852.!!

Schwartz,!J.!J.,!Huth,!K.,!Hunce,!R.,!&!Lentine,!B.!(2010).!Effect!of!anomalous!pulse!timing! on!call!discrimination!by!females!of!the!gray!treefrog!(Hyla!versicolor):!behavioral! correlates!of!neurobiology.!J#Exp#Biol,!213(12),!2066–2072.!

Seeba,!F.,!Schwartz,!J.!J.,!&!Bee,!M.!A.!(2010).!Testing!an!auditory!illusion!in!frogs:! Perceptual!restoration!or!sensory!bias?!Anim#Behav,!79(6),!1317–1328.!!

Swenson,!N.!G.!(2010).!Mapping!the!suturing!of!a!continental!biota.!Molecular#Ecology,! 19(24),!5324–7.!!

Wiley,!J.!E.,!&!Little,!M.!L.!(2000).!Replication!banding!patterns!of!the!diploid`tetraploid! treefrogs!Hyla!chrysoscelis!and!H.!versicolor.!Cytogenetics#and#Cell#Genetics,!88(1` 2),!11–14.!

!

91! ! !

VITA(

! Jennifer!Jean!Henderson!was!born!on!January!31,!1987!in!Fort!Campbell,!

Tennessee.!She!spent!the!first!few!years!of!her!life!in!Tennessee!and!New!Jersey!before! settling!in!Eagan,!Minnesota.!There,!Jennifer!graduated!from!Eastview!High!School!in!

2005!and!attended!the!University!of!Minnesota,!earning!a!Bachelor!of!Science!in!

Ecology,!Evolution,!and!Behavior!in!2008.!Jennifer!participated!in!a!number!of! undergraduate!research!projects!as!a!student!at!the!University!of!Minnesota.!Her! undergraduate!thesis!examined!male`male!interactions!in!wild!chimpanzees!as!part!of! the!Jane!Goodall!Institute!–!Center!for!Primate!Studies.!Jennifer!was!also!an!active! researcher!in!the!Animal!Communication!Lab!studying!acoustic!communication!in! treefrogs.!It!was!this!research!experience!that!ultimately!led!her!to!graduate!work!at!the!

University!of!Missouri!under!the!supervision!of!Dr.!H.!Carl!Gerhardt.!During!this!time,!

Jennifer!participated!in!numerous!science!outreach!and!science!communication! projects.!Jennifer!also!became!involved!with!broader!imapcts!at!the!national!level,! working!with!Dr.!Susan!Renoe!as!part!of!the!Broader!Imapcts!Network.!In!August!2014,!

Jennifer!returned!to!Minnesota!to!begin!working!as!the!Director!of!Education,!Outreach,! and!Diversity!at!the!Center!for!Sustainable!Polymers!at!the!University!of!Minnesota.!She! defended!her!Doctor!of!Philosophy!in!Biological!Sciences!with!minor!in!College!Teaching! in!November!2014.!

!

92! ! !